These are representative sample records from Science.gov related to your search topic.
For comprehensive and current results, perform a real-time search at Science.gov.
1

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models  

PubMed Central

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury. Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase. Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al.16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice. We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models. Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo. PMID:23117583

Han, Wei; Li, Hui; Segal, Brahm H.; Blackwell, Timothy S.

2012-01-01

2

Activity of neutrophil NADPH oxidase in iron-deficient anemia  

Microsoft Academic Search

This study was designed to measure the effects of iron supplementation on respiratory burst in iron-deficient anemia. The\\u000a performance of neutrophils was evaluated by measuring the activity of NADPH oxidase in 18 patients with iron-deficient anemia\\u000a before and after body iron stores are saturated. The activity of NADPH oxidase was significantly lower in pretreatment patients\\u000a relative to controls (p<0.05). The

Erdal Kurtoglu; Aysegul Ugur; Abdulkerim Kasim Baltaci; Rasim Mogolkoc; Levent Undar

2003-01-01

3

NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation  

PubMed Central

Neuronal NMDA receptor (NMDAR) activation leads to the formation of superoxide, which normally acts in cell signaling. With extensive NMDAR activation, the resulting superoxide production leads to neuronal death. It is widely held that NMDA-induced superoxide production originates from the mitochondria, but definitive evidence for this is lacking. We evaluated the role of the cytoplasmic enzyme NADPH oxidase in NMDA-induced superoxide production. Neurons in culture and in mouse hippocampus responded to NMDA with a rapid increase in superoxide production, followed by neuronal death. These events were blocked by the NADPH oxidase inhibitor apocynin and in neurons lacking the p47phox subunit, which is required for NADPH oxidase assembly. Superoxide production was also blocked by inhibiting the hexose monophosphate shunt, which regenerates the NADPH substrate, and by inhibiting protein kinase C zeta, which activates the NADPH oxidase complex. These findings identify NADPH oxidase as the primary source of NMDA-induced superoxide production. PMID:19503084

Brennan, Angela M; Suh, Sang Won; Won, Seok Joon; Narasimhan, Purnima; Kauppinen, Tiina M; Lee, Hokyou; Edling, Ylva; Chan, Pak H; Swanson, Raymond A

2009-01-01

4

Taurine prevents cardiomyocyte death by inhibiting NADPH oxidase-mediated calpain activation.  

PubMed

Taurine has been shown to prevent cardiomyocyte apoptosis. This study investigated the effects of taurine on NADPH oxidase and calpain activation in mediating apoptosis in cardiomyocytes. Apoptosis was induced by norepinephrine (NE) in cultured adult rat ventricular cardiomyocytes. NE (5 microM) increased NADPH oxidase activation and reactive oxygen species (ROS) production and induced apoptosis. These effects of NE on cardiomyocytes were diminished by taurine (0.5 mg/kg) but not beta-alanine. Inhibition of gp91(phox)-NADPH oxidase or ROS production protected cardiomyocytes from apoptosis. NE also induced calpain-1 activation in cardiomyocytes. This effect of NE on calpain was abrogated by gp91(phox)-NADPH oxidase inhibition or ROS scavengers and was mimicked by H(2)O(2) (25 microM) in cardiomyocytes. Pharmacological inhibitors of calpain or overexpression of calpastatin, a specific calpain inhibitor, blocked calpain activation and prevented cardiomyocyte apoptosis during NE stimulation. Furthermore, taurine treatment inhibited NE- or H(2)O(2)-induced calpain activation in cardiomyocytes. In conclusion, NADPH oxidase induces calpain activation, leading to apoptosis in NE-induced cardiomyocytes. Taurine inhibits NADPH oxidase and calpain activation. Thus, inhibition of NADPH oxidase-mediated calpain activation may be an important mechanism for taurine's antiapoptotic action in cardiomyocytes. PMID:18950702

Li, Ying; Arnold, J Malcolm O; Pampillo, Macarena; Babwah, Andy V; Peng, Tianqing

2009-01-01

5

Fungal Metabolite Gliotoxin Targets Flavocytochrome b558 in the Activation of the Human Neutrophil NADPH Oxidase  

Microsoft Academic Search

Fungal gliotoxin (GT) is a potent inhibitor of the O2 -generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47phox, a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C II with p47phox on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here,

Satoshi Nishida; Lucia S. Yoshida; Takashi Shimoyama; Hiroyuki Nunoi; Toshihiro Kobayashi; Shohko Tsunawaki

2005-01-01

6

Cholesterol: A modulator of the phagocyte NADPH oxidase activity - A cell-free study  

PubMed Central

The NADPH oxidase Nox2, a multi-subunit enzyme complex comprising membrane and cytosolic proteins, catalyzes a very intense production of superoxide ions O2•?, which are transformed into other reactive oxygen species (ROS). In vitro, it has to be activated by addition of amphiphiles like arachidonic acid (AA). It has been shown that the membrane part of phagocyte NADPH oxidase is present in lipid rafts rich in cholesterol. Cholesterol plays a significant role in the development of cardio-vascular diseases that are always accompanied by oxidative stress. Our aim was to investigate the influence of cholesterol on the activation process of NADPH oxidase. Our results clearly show that, in a cell-free system, cholesterol is not an efficient activator of NADPH oxidase like arachidonic acid (AA), however it triggers a basal low superoxide production at concentrations similar to what found in neutrophile. A higher concentration, if present during the assembly process of the enzyme, has an inhibitory role on the production of O2•?. Added cholesterol acts on both cytosolic and membrane components, leading to imperfect assembly and decreasing the affinity of cytosolic subunits to the membrane ones. Added to the cytosolic proteins, it retains their conformations but still allows some conformational change induced by AA addition, indispensable to activation of NADPH oxidase.

Masoud, Rawand; Bizouarn, Tania; Houee-Levin, Chantal

2014-01-01

7

Butylated hydroxyanisole-stimulated NADPH oxidase activity in rat liver microsomal fractions.  

PubMed

NADPH-dependent oxygen utilization by liver microsomal fractions was stimulated by the addition of increasing concentrations of butylated hydroxyanisole concomitant with the inhibition of benzphetamine N-demethylase activity. The apparent conversion of monooxygenase activity to an oxidase-like activity in the presence of the antioxidant was correlated with the partial recovery of the reducing equivalents from NADPH in the form of increased hydrogen peroxide production. The progress curve of liver microsomal NADPH oxidase activity in the presence of butylated hydroxyanisole displayed a lag phase indicative of the formation of a metabolite capable of uncoupling the monooxygenase activity. Ethyl acetate extracts of microsomal reaction mixtures obtained in the presence of butylated hydroxyanisole, oxygen, and NADPH stimulated the NADPH oxidase activity of either liver microsomes or purified NADPH-cytochrome c (P-450) reductase. Using high performance liquid chromatography, gas chromatography, and mass spectrometry techniques, two metabolites of butylated hydroxyanisole, namely t-butylhydroquinone and t-butylquinone, were identified. The quinone metabolite and/or its 1-electron reduction product interact with the flavoprotein reductase to directly link the enzyme to the reduction of oxygen which results in an inhibition of the catalytic activity of the cytochrome P-450-dependent monooxygenase. PMID:6415052

Cummings, S W; Prough, R A

1983-10-25

8

Residual NADPH Oxidase Activity and Isolated Lung Involvement in X-Linked Chronic Granulomatous Disease  

PubMed Central

Chronic granulomatous disease (CGD) is characterized by inherited immune defects resulting from mutations in the NADPH oxidase complex genes. The X-linked type of CGD is caused by defects in the CYBB gene that encodes gp91-phox, a fundamental component of the NADPH oxidase complex. This mutation originates the most common and severe form of CGD, which typically has absence of NADPH oxidase function and aggressive multisystemic infections. We present the case of a 9-year-old child with a rare CYBB mutation that preserves some NADPH oxidase activity, resulting in an atypical mild form of X-linked CGD with isolated lung involvement. Although the clinical picture and partially preserved oxidase function suggested an autosomal recessive form of CGD, genetic testing demonstrated a mutation in the exon 3 of CYBB gene (c.252 G>A, p.Ala84Ala), an uncommon X-linked CGD variant that affects splicing. Atypical presentation and diagnostic difficulties are discussed. This case highlights that the diagnosis of mild forms of X-linked CGD caused by rare CYBB mutations and partially preserved NADPH function should be considered early in the evaluation of atypical and recurrent lung infections. PMID:23193493

Gutierrez, Maria J.; McSherry, George D.; Ishmael, Faoud T.; Horwitz, Alexandra A.; Nino, Gustavo

2012-01-01

9

Rotenone induces neurotoxicity through Rac1-dependent activation of NADPH oxidase in SHSY-5Y cells.  

PubMed

Neurodegenerative diseases are attributed to impairment of the ubiquitin-proteasome system (UPS). Oxidative stress has been considered a contributing factor in the pathology of impaired UPS by promoting protein misfolding and subsequent protein aggregate formation. Increasing evidence suggests that NADPH oxidase is a likely source of excessive oxidative stress in neurodegenerative disorders. However, the mechanism of activation and its role in impaired UPS is not understood. We show that activation of NADPH oxidase in a neuroblastoma cell line (SHSY-5Y) resulted in increased oxidative and nitrosative stress, elevated cytosolic calcium, ER-stress, impaired UPS, and apoptosis. Rac1 inhibition mitigated the oxidative/nitrosative stress, prevented calcium-dependent ER-stress, and partially rescued UPS function. These findings demonstrate that Rac1 and NADPH oxidase play an important role in rotenone neurotoxicity. PMID:24374334

Pal, Rituraj; Monroe, Tanner O; Palmieri, Michela; Sardiello, Marco; Rodney, George G

2014-01-31

10

PKC? activation mediates angiogenesis via NADPH oxidase activity in PC-3 prostate cancer cells  

PubMed Central

Background PKC? is generally known as a pro-apoptotic and anti-proliferative enzyme in human prostate cancer cells. Methods Here, we investigated the role of PKC? on the growth of PC-3 human prostate cancer cells in vivo and in vitro. Results We found that sustained treatment with a specific PKC? activator (?? receptor for active C kinase, ??RACK) increased growth of PC-3 xenografts. There was increased levels of HIF-1?, vascular endothelial growth factor and CD31-positive cells in PC-3 xenografts, representative of increased tumor angiogenesis. Mechanistically, PKC? activation increased the levels of reactive oxygen species (ROS) by binding to and phosphorylating NADPH oxidase, which induced its activity. Also, PKC?-induced activation of NADPH oxidase increased the level of HIF-1?. Conclusions Our results using tumors from the PC-3 xenograft model suggest that PKC? activation increases angiogenic activity in androgen-independent PC-3 prostate cancer cells by increasing NADPH oxidase activity and HIF-1? levels and thus may partly be responsible for increased angiogenesis in advanced prostate cancer. PMID:21541971

Kim, Jeewon; Koyanagi, Tomoyoshi; Mochly-Rosen, Daria

2012-01-01

11

NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation  

Microsoft Academic Search

Neuronal NMDA receptor (NMDAR) activation leads to the formation of superoxide, which normally acts in cell signaling. With extensive NMDAR activation, the resulting superoxide production leads to neuronal death. It is widely held that NMDA-induced superoxide production originates from the mitochondria, but definitive evidence for this is lacking. We evaluated the role of the cytoplasmic enzyme NADPH oxidase in NMDA-induced

Angela M Brennan; Sang Won Suh; Seok Joon Won; Purnima Narasimhan; Tiina M Kauppinen; Hokyou Lee; Ylva Edling; Pak H Chan; Raymond A Swanson

2009-01-01

12

Regulation of innate immunity by NADPH oxidase  

PubMed Central

NADPH oxidase is a critical regulator of both antimicrobial host defense and inflammation. Activated in nature by microbes and microbial-derived products, the phagocyte NADPH oxidase is rapidly assembled, and generates reactive oxidant intermediates (ROIs) in response to infectious threat. Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by recurrent and severe bacterial and fungal infections, and pathology related to excessive inflammation. Studies in CGD patients and CGD mouse models indicate that NADPH oxidase plays a key role in modulating inflammation and injury that is distinct from its antimicrobial function. The mechanisms by which NADPH oxidase mediates killing of pathogens and regulation of inflammation has broad relevance to our understanding of normal physiological immune responses and pathological states, such as acute lung injury and bacterial or fungal infections. PMID:22583699

Segal, Brahm H.; Grimm, Melissa J.; Khan, A. Nazmul H.; Han, Wei; Blackwell, Timothy S.

2012-01-01

13

Phagocytosis-Coupled Activation of the Superoxide-Producing Phagocyte Oxidase, a Member of the NADPH Oxidase (Nox) Family  

Microsoft Academic Search

The phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a crucial role in host defense by neutrophils\\u000a and macrophages. When cells ingest invading microbes, this enzyme becomes activated to reduce molecular oxygen to superoxide,\\u000a a precursor of microbicidal oxidants, in the phagosome. The catalytic core of the oxidase is membrane-bound cytochrome b\\u000a 558, which comprises gp91\\u000a phox\\u000a and p22\\u000a phox

Reiko Minakami; Hideki Sumimotoa

2006-01-01

14

Microglial NADPH oxidase activation mediates rod cell death in the retinal degeneration in rd mice.  

PubMed

Accumulating evidence supports that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contributes to microglia-mediated neurotoxicity in the CNS neurodegenerative diseases. Several studies, including ours, suggest that microglial activation is involved in the retinal degeneration in the animal models of retinitis pigmentosa (RP). In the present study, we investigated the activation of NADPH oxidase in the rod degeneration in rd mice and further explored its role in the microglia-mediated photoreceptor apoptosis. Expression of gp91phox protein, a major subunit of NAPDH oxidase in the whole retina of rd mice at postnatal days (P) 8, 10, 12, 14, 16 and 18 was assessed by western blot analysis. Location of gp91phox in the rd retina at each age group and its cellular source were studied by immunohistochemical analysis and double labeling respectively. The generation of superoxide radicals in the rd retinas was demonstrated by intraperitoneal injection of hydroethidine. Apocynin was applied intraperitoneally in the rd mice from P8 to P14 to inhibit the activity of NAPDH oxidase and the outer nuclear layer (ONL) thickness was measured before and after apocynin treatment. Our results demonstrated that during the rod degenerative process, the expression of gp91phox started to increase in the outer part of rd retina at P10 and reached a peak at P14. Double labeling of gp91phox with CD11b showed co-localization of gp91phox in the retinal microglial cells. Increasing generation of superoxide radicals visualized by hydroethidine was noted at P8 and reached a peak at P14. Apocynin markedly reduced the production of superoxide radicals and preserved the rod cells. The results suggested that NADPH oxidase might play an important role in the rod degeneration in the rd mice. Inhibition of NAPDH oxidase could be a possible approach to treat RP in the early degenerative stage. PMID:24929065

Zeng, H; Ding, M; Chen, X-X; Lu, Q

2014-09-01

15

Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils  

PubMed Central

Generation of reactive oxygen species by the NADPH oxidase complex is an important bactericidal weapon of phagocytes. Phorbol myristate acetate (PMA) is a potent agonist for this “respiratory burst” in human neutrophils. Although stoichiometric H+ efflux occurs during the respiratory burst, efforts to stimulate voltage-gated H+ channels by PMA in whole-cell patch-clamped phagocytes have been unsuccessful. We have used a modification of the permeabilized-patch configuration that allows control of intracellular pH and preserves second-messenger pathways. Using this method, we show that PMA dramatically enhances and alters voltage-gated proton currents in human neutrophils. PMA produced four alterations in H+ current properties, each of which increases the H+ current at any given voltage: (i) a 40-mV negative shift in the H+ conductance-voltage (gH–V) relationship; (ii) faster activation [smaller activation time constant (?act)] during depolarizing pulses; (iii) slower deactivation [larger deactivation time constant (?tail)] on repolarization; and (iv) a larger maximum H+ conductance (gH,max). Inward current that directly reflects electron transport by NADPH oxidase was also activated by PMA stimulation. The identity of this electron current was confirmed by its sensitivity to diphenylene iodinium, an inhibitor of NADPH oxidase. Diphenylene iodinium also reversed the slowing of ?tail with a time course paralleling the inhibition of electron current. However, the amplitudes of H+ and electron currents activated by PMA were not correlated. A complex interaction between NADPH oxidase and voltage-gated proton channels is indicated. The data suggest that PMA stimulation modulates preexisting H+ channels rather than inducing a new H+ channel. PMID:10823889

DeCoursey, T. E.; Cherny, V. V.; Zhou, W.; Thomas, L. L.

2000-01-01

16

Curcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils.  

PubMed

In neutrophils (PMNs), superoxide anion (O2*-), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation of cytosolic subunits to the membrane flavocytochrome b558. In horses, excessive activation of PMNs is often associated with deadly pathologies and the modulation of their ROS production by acting on NADPH oxidase is a prime target to manage inflammation. We developed a cell-free assay to measure the activity of equine NADPH oxidase assembled in vitro, in order to test the effects of natural or synthetic compounds on the enzyme activity or assembly. The cell-free assay was validated with diphenyleneiodonium chloride and Gp91ds-tat, two inhibitors largely described for human NADPH oxidase. The anti-oxidant effects of curcumin and resveratrol at final concentration ranging from 10(-4) to 10(-6) M were studied on whole cells by chemiluminescence (CL) and by cell-free assay, in which the molecule was added before or after the enzyme assembly. The CL assay demonstrated that curcumin efficiently inhibited the O2(-) production and easily entered into PMNs or interacted with their membrane. Cell-free assay showed that curcumin acted on the reconstitution of NADPH oxidase even at 10(-5)M, while resveratrol appeared to be an O2*- scavenger rather than an inhibitor of NADPH oxidase activity, since it acted from outside the cell in CL and after the complex assembly in cell-free assay. By acting directly on NADPH oxidase, curcumin should be a good candidate for the treatment of acute or inflammatory diseases involving an excessive ROS production. PMID:24060679

Derochette, Sandrine; Franck, Thierry; Mouithys-Mickalad, Ange; Ceusters, Justine; Deby-Dupont, Ginette; Lejeune, Jean-Philippe; Neven, Philippe; Serteyn, Didier

2013-11-25

17

NOX2?: A Novel Splice Variant of NOX2 That Regulates NADPH Oxidase Activity in Macrophages  

PubMed Central

Nox2 oxidase is one isoform in a family of seven NADPH oxidases that generate reactive oxygen species (ROS) and thereby contribute to physiological and pathological processes including host defense, redox signaling and oxidative tissue damage. While alternative mRNA splicing has been shown to influence the activity of several Nox-family proteins, functionally relevant splice variants of Nox2 have not previously been identified. We immunoscreened several mouse tissues and cells for the presence of truncated Nox2 proteins and identified a 30 kDa protein in lung, spleen and macrophages. RT-PCR analysis of mRNA from primary and immortalised (RAW264.7) mouse macrophages, and from human alveolar macrophages, identified a truncated Nox2 transcript which, upon sequence analysis, was found to be a product of the ‘exon skipping’ mode of alternative splicing, lacking exons 4–10 of the Nox2 gene. The predicted protein is comparable in size to that identified by immunoscreening and contains two transmembrane helices and an extended cytosolic C-terminus with binding sites for NADPH and the Nox organiser protein p47phox. Importantly, selective siRNA-mediated knockdown of the transcript reduced expression of the 30 kDa protein in macrophages, and suppressed phorbol ester-stimulated ROS production by 50%. We thus provide the first evidence that Nox2 undergoes alternative mRNA splicing to yield a 30 kDa protein – herein termed Nox2? – that regulates NADPH oxidase activity in macrophages from mice and humans. The discovery of Nox2? paves the way for future examination of its role in physiological and pathological processes. PMID:23118986

Guida, Elizabeth; King, Paul T.; Sobey, Christopher G.; Drummond, Grant R.

2012-01-01

18

NADPH oxidase: a universal oxygen sensor?  

Microsoft Academic Search

NADPH oxidase is classically regarded as a key enzyme of neutrophils, where it is involved in the pathogenic production of reactive oxygen species. However, NADPH oxidase-like enzymes have recently been identified in non-neutrophil cells, supporting a separate role for NADPH-oxidase derived oxygen species in oxygen sensitive processes. This article reviews the current literature surrounding the potential role of NADPH oxidase

Richard D Jones; John T Hancock; Alyn H Morice

2000-01-01

19

A Role for NADPH Oxidase in Antigen Presentation  

PubMed Central

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expressed in phagocytes is a multi-subunit enzyme complex that generates superoxide (O2.?). This radical is an important precursor of hydrogen peroxide (H2O2) and other reactive oxygen species needed for microbicidal activity during innate immune responses. Inherited defects in NADPH oxidase give rise to chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent infections and granulomatous inflammation. Interestingly, CGD, CGD carrier status, and oxidase gene polymorphisms have all been associated with autoinflammatory and autoimmune disorders, suggesting a potential role for NADPH oxidase in regulating adaptive immune responses. Here, NADPH oxidase function in antigen processing and presentation is reviewed. NADPH oxidase influences dendritic cell (DC) crosspresentation by major histocompatibility complex class I molecules through regulation of the phagosomal microenvironment, while in B lymphocytes, NADPH oxidase alters epitope selection by major histocompatibility complex class II molecules. PMID:24069023

Gardiner, Gail J.; Deffit, Sarah N.; McLetchie, Shawna; Perez, Liliana; Walline, Crystal C.; Blum, Janice S.

2013-01-01

20

Vitamins reverse endothelial dysfunction through regulation of eNOS and NAD(P)H oxidase activities.  

PubMed

Antioxidant vitamins C and E have protective properties in genetic hypertension associated with enhanced oxidative stress. This study investigated whether vitamins C and/or E modulate vascular function by regulating enzymatic activities of endothelial nitric oxide synthase (eNOS) and NAD(P)H oxidase using thoracic aortas of 20- to 22-week-old male spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar-Kyoto rats (WKY). SHR aortas had impaired relaxant responses to acetylcholine but not to sodium nitroprusside, despite an approximately 2-fold increase in eNOS activity and NO release. The levels of superoxide anion (O2-), a potent NO scavenger, and NAD(P)H oxidase activity were also 2-fold higher in SHR aortas. Mechanical but not pharmacological inactivation of endothelium (by rubbing and 100 micromol/L L-NAME, respectively) significantly abrogated O2- in both strains. Treatments of SHR aortas with NAD(P)H oxidase inhibitors, namely diphenyleneiodinium and apocynin, significantly diminished O2- production. The incubation of SHR aortas with different concentrations of vitamin C (10 to 100 micromol/L) and specifically with high concentrations of vitamin E (100 micromol/L) improved endothelial function, reduced superoxide production as well as NAD(P)H oxidase activity, and increased eNOS activity and NO generation in SHR aortas to the levels observed in vitamin C- and E-treated WKY aortas. Our results reveal endothelial NAD(P)H oxidase as the major source of vascular O2- in SHR and also show that vitamins C and E are critical in normalizing genetic endothelial dysfunction through regulation of eNOS and NAD(P)H oxidase activities. PMID:12623955

Ulker, Sibel; McKeown, Pascal P; Bayraktutan, Ulvi

2003-03-01

21

Targeting NADPH oxidases in vascular pharmacology  

PubMed Central

Oxidative stress is a molecular dysregulation in reactive oxygen species (ROS) metabolism, which plays a key role in the pathogenesis of atherosclerosis, vascular inflammation and endothelial dysfunction. It is characterized by a loss of nitric oxide (NO) bioavailability. Large clinical trials such as HOPE and HPS have not shown a clinical benefit of antioxidant vitamin C or vitamin E treatment, putting into question the role of oxidative stress in cardiovascular disease. A change in the understanding of the molecular nature of oxidative stress has been driven by the results of these trials. Oxidative stress is no longer perceived as a simple imbalance between the production and scavenging of ROS, but as a dysfunction of enzymes involved in ROS production. NADPH oxidases are at the center of these events, underlying the dysfunction of other oxidases including eNOS uncoupling, xanthine oxidase and mitochondrial dysfunction. Thus NADPH oxidases are important therapeutic targets. Indeed, HMG-CoA reductase inhibitors (statins) as well as drugs interfering with the renin-angiotensin-aldosterone system inhibit NADPH oxidase activation and expression. Angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists (sartans) and aliskiren, as well as spironolactone or eplerenone, have been discussed. Molecular aspects of NADPH oxidase regulation must be considered, while thinking about novel pharmacological targeting of this family of enzymes consisting of several homologs Nox1, Nox2, Nox3, Nox4 and Nox5 in humans. In order to properly design trials of antioxidant therapies, we must develop reliable techniques for the assessment of local and systemic oxidative stress. Classical antioxidants could be combined with novel oxidase inhibitors. In this review, we discuss NADPH oxidase inhibitors such as VAS2870, VAS3947, GK-136901, S17834 or plumbagin. Therefore, our efforts must focus on generating small molecular weight inhibitors of NADPH oxidases, allowing the selective inhibition of dysfunctional NADPH oxidase homologs. This appears to be the most reasonable approach, potentially much more efficient than non-selective scavenging of all ROS by the administration of antioxidants. PMID:22405985

Schramm, Agata; Matusik, Pawel; Osmenda, Grzegorz; Guzik, Tomasz J

2012-01-01

22

Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity.  

PubMed Central

VCAM-1 (vascular cell adhesion molecule-1) plays an important role in the regulation of inflammation in atherosclerosis, asthma, inflammatory bowel disease and transplantation. VCAM-1 activates endothelial cell NADPH oxidase, and this oxidase activity is required for VCAM-1-dependent lymphocyte migration. We reported previously that a mouse microvascular endothelial cell line promotes lymphocyte migration that is dependent on VCAM-1, but not on other known adhesion molecules. Here we have investigated the signalling mechanisms underlying VCAM-1 function. Lymphocyte binding to VCAM-1 on the endothelial cell surface activated an endothelial cell calcium flux that could be inhibited with anti-alpha4-integrin and mimicked by anti-VCAM-1-coated beads. VCAM-1 stimulation of calcium responses could be blocked by an inhibitor of intracellular calcium mobilization, a calcium channel inhibitor or a calcium chelator, resulting in the inhibition of NADPH oxidase activity. Addition of ionomycin overcame the calcium channel blocker suppression of VCAM-1-stimulated NADPH oxidase activity, but could not reverse the inhibitory effect imposed by intracellular calcium blockage, indicating that both intracellular and extracellular calcium mobilization are required for VCAM-1-mediated activation of NADPH oxidase. Furthermore, VCAM-1 specifically activated the Rho-family GTPase Rac1, and VCAM-1 activation of NADPH oxidase was blocked by a dominant negative Rac1. Thus VCAM-1 stimulates the mobilization of intracellular and extracellular calcium and Rac1 activity that are required for the activation of NADPH oxidase. PMID:14594451

Cook-Mills, Joan M; Johnson, Jacob D; Deem, Tracy L; Ochi, Atsuo; Wang, Lei; Zheng, Yi

2004-01-01

23

Tumor necrosis factor-?-induced nuclear factor-kappaB activation in human cardiomyocytes is mediated by NADPH oxidase.  

PubMed

An elevated level of tumor necrosis factor (TNF)-? is implicated in several cardiovascular diseases including heart failure. Numerous reports have demonstrated that TNF-? activates nuclear factor (NF)-kappaB, resulting in the upregulation of several genes that regulate inflammation, proliferation, and apoptosis of cardiomyocytes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of reactive oxygen species (ROS), is also activated by TNF-? and plays a crucial role in redox-sensitive signaling pathways. The present study investigated whether NADPH oxidase mediates TNF-?-induced NF-kappaB activation and NF-kappaB-mediated gene expression. Human cardiomyocytes were treated with recombinant TNF-? with or without pretreatment with diphenyleneiodonium (DPI) and apocynin, inhibitors of NADPH oxidase. TNF-?-induced ROS production was measured using 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate assay. TNF-?-induced NF-kappaB activation was also examined using immunoblot; NF-kappaB binding to its binding motif was determined using a Cignal reporter luciferase assay and an electrophoretic mobility shift assay. TNF-?-induced upregulation of interleukin (IL)-1? and vascular cell adhesion molecule (VCAM)-1 was investigated using real-time PCR and immunoblot. TNF-?-induced ROS production in cardiomyocytes was mediated by NADPH oxidase. Phosphorylation of IKK-?/? and p65, degradation of IkappaB?, binding of NF-kappaB to its binding motif, and upregulation of IL-1? and VCAM-1 induced by TNF-? were significantly attenuated by treatment with DPI and apocynin. Collectively, these findings demonstrate that NADPH oxidase plays a role in regulation of TNF-?-induced NF-kappaB activation and upregulation of proinflammatory cytokines, IL-1? and VCAM-1, in human cardiomyocytes. PMID:25059721

Moe, Kyaw Thu; Khairunnisa, Katwadi; Yin, Nwe Oo; Chin-Dusting, Jaye; Wong, Philip; Wong, Meng Cheong

2014-09-01

24

The complex roles of NADPH oxidases in fungal infection.  

PubMed

NADPH oxidases play key roles in immunity and inflammation that go beyond the production of microbicidal reactive oxygen species (ROS). The past decade has brought a new appreciation for the diversity of roles played by ROS in signalling associated with inflammation and immunity. NADPH oxidase activity affects disease outcome during infections by human pathogenic fungi, an important group of emerging and opportunistic pathogens that includes Candida, Aspergillus and Cryptococcus species. Here we review how alternative roles of NADPH oxidase activity impact fungal infection and how ROS signalling affects fungal physiology. Particular attention is paid to roles for NADPH oxidase in immune migration, immunoregulation in pulmonary infection, neutrophil extracellular trap formation, autophagy and inflammasome activity. These recent advances highlight the power and versatility of spatiotemporally controlled redox regulation in the context of infection, and point to a need to understand the molecular consequences of NADPH oxidase activity in the cell. PMID:24905433

Hogan, Deborah; Wheeler, Robert T

2014-08-01

25

Diacylglycerol kinases terminate diacylglycerol signaling during the respiratory burst leading to heterogeneous phagosomal NADPH oxidase activation.  

PubMed

It is commonly assumed that all phagosomes have identical molecular composition. This assumption has remained largely unchallenged due to a paucity of methods to distinguish individual phagosomes. We devised an assay that extends the utility of nitro blue tetrazolium for detection and quantification of NAPDH oxidase (NOX) activity in individual phagosomes. Implementation of this assay revealed that in murine macrophages there is heterogeneity in the ability of individual phagosomes to generate superoxide, both between and within cells. To elucidate the molecular basis of the variability in NOX activation, we employed genetically encoded fluorescent biosensors to evaluate the uniformity in the distribution of phospholipid mediators of the oxidative response. Despite variability in superoxide generation, the distribution of phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-phosphate, and phosphatidic acid was nearly identical in all phagosomes. In contrast, diacylglycerol (DAG) was not generated uniformly across the phagosomal population, varying in a manner that directly mirrored superoxide production. Modulation of DAG levels suggested that NOX activation is precluded when phagosomes fail to reach a critical DAG concentration. In particular, forced expression of diacylglycerol kinase ? abrogated DAG accumulation at the phagosome, leading to impaired respiratory burst. Conversely, pharmacological inhibition of DAG kinases or expression of an inactive diacylglycerol kinase ? mutant increased the proportion of DAG-positive phagosomes, concomitantly potentiating phagosomal NOX activity. Our data suggest that diacylglycerol kinases limit the extent of NADPH oxidase activation, curtailing the production of potentially harmful reactive oxygen species. The resulting heterogeneity in phagosome responsiveness could enable the survival of a fraction of invading microorganisms. PMID:23814057

Schlam, Daniel; Bohdanowicz, Michal; Chatgilialoglu, Alexandros; Chatilialoglu, Alexandros; Steinberg, Benjamin E; Ueyama, Takehiko; Du, Guangwei; Grinstein, Sergio; Fairn, Gregory D

2013-08-01

26

NADPH oxidase (NOX2) activity is a modifier of survival in ALS.  

PubMed

NADPH-oxidases (NOX) catalyze the formation of reactive oxygen species (ROS), which play a role in the development of neurological diseases, particularly those generated by the phagocytic isoform NOX2. Increased ROS has been observed in the amyotrophic lateral sclerosis (ALS) SOD1 transgenic mouse, and in this preclinical model the inactivation of NOX2 decreases ROS production and extends survival. Our aim was to evaluate NOX2 activity measuring neutrophil oxidative burst in a cohort of 83 ALS patients, and age- and gender-matched healthy controls. Oxidative burst was measured directly in fresh blood using Phagoburst™ assay by flow cytometry. Mean fluorescence intensity (MFI), emitted in response to different stimuli, leads to produce ROS and corresponds to the percentage of oxidizing cells and their enzymatic activity (GeoMean). No difference was found between the MFI values in cases and controls. NOX2 activity was independent from gender and age, and in patients was not related to disease duration, site of onset (bulbar vs. spinal), or ALSFRS-R score. However, patients with a NOX2 activity lower than the median value showed a 1-year increase of survival from onset (p = 0.011). The effect of NOX2 was independent from other known prognostic factors. These findings are in keeping with the observations in the mouse model of ALS, and demonstrate the strong role of NOX2 in modifying progression in ALS patients. A proper modulation of NOX2 activity might hold therapeutic potential for ALS. PMID:25178511

Marrali, Giuseppe; Casale, Federico; Salamone, Paolina; Fuda, Giuseppe; Caorsi, Cristiana; Amoroso, Antonio; Brunetti, Maura; Restagno, Gabriella; Barberis, Marco; Bertuzzo, Davide; Canosa, Antonio; Moglia, Cristina; Calvo, Andrea; Chiň, Adriano

2014-11-01

27

NMDA Receptor-Mediated Activation of NADPH Oxidase and Glomerulosclerosis in Hyperhomocysteinemic Rats  

PubMed Central

Abstract This study investigated the role of NMDA receptor in hyperhomocyteinemia (hHcys)-induced NADPH oxidase (Nox) activation and glomerulosclerosis. Sprague–Dawley rats were fed a folate-free (FF) diet to produce hHcys, and a NMDA receptor antagonist, MK-801, was administrated. Rats fed the FF diet exhibited significantly increased plasma homocysteine levels, upregulated NMDA receptor expression, enhanced Nox activity and Nox-dependent O2.? production in the glomeruli, which were accompanied by remarkable glomerulosclerosis. MK-801 treatment significantly inhibited Nox-dependent O2.? production induced by hHcys and reduced glomerular damage index as compared with vehicle-treated hHcys rats. Correspondingly, glomerular deposition of extracellular matrix components in hHcys rats was ameliorated by the administration of MK-801. Additionally, hHcys induced an increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) expression and a decrease in matrix metalloproteinase (MMP)-1 and MMP-9 activities, all of which were abolished by MK-801 treatment. In vitro studies showed that homocysteine increased Nox-dependent O2.? generation in rat mesangial cells, which was blocked by MK-801. Pretreatment with MK-801 also reversed homocysteine-induced decrease in MMP-1 activity and increase in TIMP-1 expression. These results support the view that the NMDA receptor may mediate Nox activation in the kidney during hHcys and thereby play a critical role in the development of hHcys-induced glomerulosclerosis. Antioxid. Redox Signal. 13, 975–986. PMID:20406136

Zhang, Chun; Yi, Fan; Xia, Min; Boini, Krishna M.; Zhu, Qing; Laperle, Laura A.; Abais, Justine M.; Brimson, Christopher A.

2010-01-01

28

Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes.  

PubMed

Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47(phox) phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress. PMID:21651898

Kasi, Viswanath; Bodiga, Sreedhar; Kommuguri, Upendra Nadh; Sankuru, Suneetha; Bodiga, Vijaya Lakshmi

2011-07-01

29

Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor  

NASA Technical Reports Server (NTRS)

The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

Morre, D. James

2002-01-01

30

Natural Compounds as Modulators of NADPH Oxidases  

PubMed Central

Reactive oxygen species (ROS) are cellular signals generated ubiquitously by all mammalian cells, but their relative unbalance triggers also diseases through intracellular damage to DNA, RNA, proteins, and lipids. NADPH oxidases (NOX) are the only known enzyme family with the sole function to produce ROS. The NOX physiological functions concern host defence, cellular signaling, regulation of gene expression, and cell differentiation. On the other hand, increased NOX activity contributes to a wide range of pathological processes, including cardiovascular diseases, neurodegeneration, organ failure, and cancer. Therefore targeting these enzymatic ROS sources by natural compounds, without affecting the physiological redox state, may be an important tool. This review summarizes the current state of knowledge of the role of NOX enzymes in physiology and pathology and provides an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols. PMID:24381714

2013-01-01

31

NADPH oxidase limits lipopolysaccharide-induced lung inflammation and injury in mice through redox regulation of NF-?B activity  

PubMed Central

Although reactive oxygen species (ROS) produced by NADPH oxidase are known to regulate inflammatory responses, the impact of ROS on intracellular signaling pathways is incompletely understood. In these studies, we treated wild-type and p47phox deficient mice with LPS to investigate mechanisms by which NADPH oxidase regulates signaling through the NF-?B pathway. After intratracheal instillation of LPS, ROS generation was impaired in p47phox-/- mice while these mice had increased neutrophilic alveolitis and greater lung injury compared to wild-type controls. In mice interbred with transgenic NF-?B reporters (HLL), we found exaggerated LPS-induced NF-?B activation and increased expression of pro-inflammatory cytokines in lungs of p47phox-/-/HLL mice compared to controls. Both lung macrophages and bone marrow-derived macrophages (BMDMs) isolated from p47phox-/-/HLL mice showed enhanced LPS-stimulated NF-?B activity compared to controls. While nuclear translocation of NF-?B proteins was similar between genotypes, electrophoretic mobility shift assays under non-reducing conditions showed increased DNA binding in p47phox-/-/HLL BMDMs, suggesting that ROS production reduces NF-?B binding to DNA without affecting nuclear translocation. Increased intracellular GSH/GSSG ratio and greater nuclear redox factor 1 (Ref-1) levels were present in p47phox-/-/HLL compared to wild-type BMDMs, pointing to NADPH oxidase modulating intracellular redox status in macrophages. Treatment with the Ref-1 specific inhibitor E3330 or hydrogen peroxide inhibited LPS-induced NF-?B activation in p47phox-/-/HLL BMDMs but not in wild-type/HLL cells. Consistent with these findings, siRNA against Ref-1 selectively reduced NF-?B activity in LPS-treated p47phox-/-/HLL BMDMs. Together, these results indicate that NADPH oxidase limits LPS-induced NF-?B transcriptional activity through regulation of intracellular redox state. PMID:23530143

Han, Wei; Li, Hui; Cai, Jiyang; Gleaves, Linda A.; Polosukhin, Vasiliy V.; Segal, Brahm H.; Yull, Fiona E.; Blackwell, Timothy S.

2013-01-01

32

Induction of genes encoding NADPH oxidase components and activation of IFN regulatory factor-1 by prolactin in fish macrophages.  

PubMed

The role played by prolactin (PRL) in fish immunity is scant. We report here that stimulation of the Atlantic salmon monocytic cell line SHK-1 with native salmon PRL resulted in activation of the respiratory burst and induction of the expression of the genes encoding the phagocyte NADPH oxidase components p47phox, p67phox and gp91phox, and the transcription factor IFN regulatory factor-1 (IRF-1). Interestingly, the pharmacologic inhibition of the Jak/Stat signaling pathway with AG490 blocked reactive oxygen species (ROS) production, and the induction of genes encoding the NADPH oxidase components and IRF-1 in PRL-activated SHK-1 cells. In addition, PRL promoted the phosphorylation of Stat and induced the DNA binding activity of IRF-1. These results, together with the presence of several consensus target motifs for Stat and IRF-1 in the promoter of the tilapia p47phox gene, suggest that PRL regulates p47phox gene expression in fish through the activation of these two key transcription factors. Taken together, our results demonstrate that PRL induces the expression of the genes encoding the major phagocyte NADPH oxidase components and ROS production in fish macrophages via the JAK2/Stat/IRF-1 signaling pathway. PMID:23548829

Olavarría, Víctor H; Figueroa, Jaime E; Mulero, Victoriano

2013-12-01

33

Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat  

Microsoft Academic Search

BackgroundRecent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in

Limor Raz; Quan-Guang Zhang; Cai-Feng Zhou; Dong Han; Priya Gulati; Li-Cai Yang; Fang Yang; Rui-Min Wang; Darrell W. Brann; Mark R. Cookson

2010-01-01

34

Elevated NADPH oxidase activity contributes to oxidative stress and cell death in Huntington's disease  

PubMed Central

A mutation in the huntingtin (Htt) gene produces mutant Htt and Huntington's disease (HD), a neurodegenerative disorder. HD patients have oxidative damage in the brain, but the causes are unclear. Compared with controls, we found brain levels of NADPH oxidase (NOX) activity, which produces reactive oxygen species (ROS), elevated in human HD postmortem cortex and striatum and highest in striatum of presymptomatic individuals. Synaptosome fractions from cortex and striatum of HD140Q/140Q mice had elevated NOX activity at 3 months of age and a further rise at 6 and 12 months compared with synaptosomes of age-matched wild-type (WT) mice. High NOX activity in primary cortical and striatal neurons of HD140Q/140Q mice correlated with more ROS and neurite swellings. These features and neuronal cell death were markedly reduced by treatment with NOX inhibitors such as diphenyleneiodonium (DPI), apocynin (APO) and VAS2870. The rise in ROS levels in mitochondria of HD140Q/140Q neurons followed the rise in NOX activity and inhibiting only mitochondrial ROS was not neuroprotective. Mutant Htt colocalized at plasma membrane lipid rafts with gp91-phox, a catalytic subunit for the NOX2 isoform. Assembly of NOX2 components at lipid rafts requires activation of Rac1 which was also elevated in HD140Q/140Q neurons. HD140Q/140Q mice bred to gp91-phox knock-out mice had lower NOX activity in the brain and in primary neurons, and neurons had normal ROS levels and significantly improved survival. These findings suggest that increased NOX2 activity at lipid rafts is an early and major source of oxidative stress and cell death in HD140Q/140Q neurons. PMID:23223017

Valencia, Antonio; Sapp, Ellen; Kimm, Jeffrey S.; McClory, Hollis; Reeves, Patrick B.; Alexander, Jonathan; Ansong, Kwadwo A.; Masso, Nicholas; Frosch, Matthew P.; Kegel, Kimberly B.; Li, Xueyi; DiFiglia, Marian

2013-01-01

35

NADPH oxidase and aging drive microglial activation, oxidative stress, and dopaminergic neurodegeneration following systemic LPS administration.  

PubMed

Parkinson's disease is characterized by a progressive degeneration of substantia nigra (SN) dopaminergic neurons with age. We previously found that a single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) injection caused a slow progressive loss of tyrosine hydroxylase immunoreactive (TH+IR) neurons in SN associated with increasing motor dysfunction. In this study, we investigated the role of NADPH oxidase (NOX) in inflammation-mediated SN neurotoxicity. A comparison of control (NOX2(+/+) ) mice with NOX subunit gp91(phox) -deficient (NOX2(-/-) ) mice 10 months after LPS administration (5 mg/kg, i.p.) resulted in a 39% (P < 0.01) loss of TH+IR neurons in NOX2(+/+) mice, whereas NOX2(-/-) mice did not show a significant decrease. Microglia (Iba1+IR) showed morphological activation in NOX2(+/+) mice, but not in NOX2(-/-) mice at 1 hr. Treatment of NOX2(+/+) mice with LPS resulted in a 12-fold increase in NOX2 mRNA in midbrain and 5.5-6.5-fold increases in NOX2 protein (+IR) in SN compared with the saline controls. Brain reactive oxygen species (ROS), determined using diphenyliodonium histochemistry, was increased by LPS in SN between 1 hr and 20 months. Diphenyliodonium (DPI), an NOX inhibitor, blocked LPS-induced activation of microglia and production of ROS, TNF?, IL-1?, and MCP-1. Although LPS increased microglial activation and ROS at all ages studied, saline control NOX2(+/+) mice showed age-related increases in microglial activation, NOX, and ROS levels at 12 and 22 months of age. Together, these results suggest that NOX contributes to persistent microglial activation, ROS production, and dopaminergic neurodegeneration that persist and continue to increase with age. PMID:23536230

Qin, Liya; Liu, Yuxin; Hong, Jau-Shyong; Crews, Fulton T

2013-06-01

36

A role for NADPH oxidase 4 in the activation of vascular endothelial cells by oxidized phospholipids.  

PubMed

Previous studies from our group have demonstrated that oxidized 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC) activates over 1000 genes in human aortic endothelial cells (HAECs). Prominent among these are genes regulating inflammation, cholesterol homeostasis, antioxidant enzymes, and the unfolded protein response. Previous studies from our lab and others suggested that transcriptional regulation by Ox-PAPC may be controlled, at least in part, by reactive oxygen species. We now present evidence that Ox-PAPC activation of NADPH oxidase 4 (NOX4) is responsible for the regulation of two of these important groups of genes: those controlling inflammation and those involved in sterol regulation. Our data demonstrate that Ox-PAPC increases reactive oxygen species formation in HAECs as seen by DCF fluorescence. NOX4 is the major molecule responsible for this increase because downregulation of NOX4 and its components (p22(phox) and rac1) blocked the Ox-PAPC effect. Our data show that Ox-PAPC did not change NOX4 transcription levels but did induce recruitment of rac1 to the membrane for NOX4 activation. We present evidence that vascular endothelial growth factor receptor 2 (VEGFR2) activation is responsible for rac1 recruitment to the membrane. Finally, we demonstrate that knockdown of NOX4 and its components rac1 and p22(phox) decreases Ox-PAPC induction of inflammatory and sterol regulatory genes, but does not affect Ox-PAPC transcriptional regulation of other genes for antioxidants and the unfolded protein response. In summary, we have identified a VEGFR2/NOX4 regulatory pathway by which Ox-PAPC controls important endothelial functions. PMID:19375500

Lee, Sangderk; Gharavi, Nima M; Honda, Henry; Chang, Irene; Kim, Brandon; Jen, Nelson; Li, Rongsong; Zimman, Alejandro; Berliner, Judith A

2009-07-15

37

The NADPH oxidase inhibitor diphenyleneiodonium activates the human TRPA1 nociceptor.  

PubMed

Transient receptor potential ankyrin 1 (TRPA1) is a Ca(2+)-permeable nonselective cation channel expressed in neuronal and nonneuronal cells and plays an important role in acute and inflammatory pain. Here, we show that an NADPH oxidase (NOX) inhibitor, diphenyleneiodonium (DPI), functions as a TRPA1 activator in human embryonic kidney cells expressing human TRPA1 (HEK-TRPA1) and in human fibroblast-like synoviocytes. Application of DPI at 0.03-10 ?M induced a Ca(2+) response in HEK-TRPA1 cells in a concentration-dependent manner. The Ca(2+) response was effectively blocked by a selective TRPA1 antagonist, HC-030031 (HC). In contrast, DPI had no effect on HEK cells expressing TRPV1-V4 or TRPM8. Four other NOX inhibitors, apocynin (APO), VAS2870 (VAS), plumbagin, and 2-acetylphenothiazine, also induced a Ca(2+) response in HEK-TRPA1 cells, which was inhibited by pretreatment with HC. In the presence of 5 mM glutathione, the Ca(2+) response to DPI was effectively reduced. Moreover, mutation of cysteine 621 in TRPA1 substantially inhibited the DPI-induced Ca(2+) response, while it did not inhibit the APO- and VAS-induced responses. The channel activity was induced by DPI in excised membrane patches with both outside-out and inside-out configurations. Internal application of neomycin significantly inhibited the DPI-induced inward currents. In inflammatory synoviocytes with TRPA1, DPI evoked a Ca(2+) response that was sensitive to HC. In mice, intraplantar injection of DPI caused a pain-related response which was inhibited by preadministration with HC. Taken together, our findings demonstrate that DPI and other NOX inhibitors activate human TRPA1 without mediating NOX. PMID:24944203

Suzuki, Hiroka; Hatano, Noriyuki; Muraki, Yukiko; Itoh, Yuka; Kimura, Satoko; Hayashi, Hidetoshi; Onozaki, Kikuo; Ohi, Yoshiaki; Haji, Akira; Muraki, Katsuhiko

2014-08-15

38

Epithelial-to-Mesenchymal Transition in Podocytes Mediated by Activation of NADPH Oxidase in Hyperhomocysteinemia  

PubMed Central

The present study tested the hypothesis that hyperhomocysteinemia (hHcys) induces podocytes to undergo epithelial-to-mesenchymal transition (EMT) through the activation of NADPH oxidase (Nox). It was found that increased homocysteine (Hcys) level suppressed the expression of slit diaphragm-associated proteins, P-cadherin and zonula occludens-1 (ZO-1) in conditionally immortalized mouse podocytes, indicating the loss of their epithelial features. Meanwhile, Hcys remarkably increased the abundance of mesenchymal markers, such as fibroblast specific protein-1 (FSP-1) and ?-smooth muscle actin (?-SMA). These phenotype changes in podocytes induced by Hcys were accompanied by enhanced superoxide (O2.?) production, which was substantially suppressed by inhibition of Nox activity. Functionally, Hcys significantly enhanced the permeability of the podocyte monolayer coupled with increased EMT, and this EMT-related increase in cell permeability could be restored by Nox inhibitors. In mice lacking gp91phox (gp91?/?), an essential Nox subunit gene, hHcys-enhanced podocyte EMT and consequent glomerular injury were examined. In wild-type (gp91+/+) mice, hHcys induced by a folate-free (FF) diet markedly enhanced expression of mesenchymal markers (FSP-1 and ?-SMA) but decreased expression of epithelial markers of podocytes in glomeruli, which were not observed in gp91?/? mouse glomeruli. Podocyte injury, glomerular sclerotic pathology, and marked albuminuria observed in gp91+/+ mice with hHcys were all significantly attenuated in gp91?/? mice. These results suggest that hHcys induces EMT of podocytes through activation of Nox, which represents a novel mechanism of hHcys-associated podocyte injury. PMID:21647593

Zhang, Chun; Xia, Min; Boini, Krishna M.; Li, Cai-Xia; Abais, Justine M.; Li, Xiao-Xue; Laperle, Laura A.; Li, Pin-Lan

2012-01-01

39

NADPH-oxidase 2 activation promotes opioid-induced antinociceptive tolerance in mice  

PubMed Central

The analgesic effectiveness of long-term opioid therapies is compromised by the development of antinociceptive tolerance linked to the overt production of peroxynitrite (ONOO?, PN), the product of the interaction between superoxide (O2??, SO) and nitric oxide (NO), and to neuroinflammatory processes. We have recently reported that in addition to post-translational nitration and inactivation of mitochondrial MnSOD, activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase holoenzyme (NOX) in the spinal cord is a major source for the overt production of superoxide-derived PN during the development of morphine-induced antinociceptive tolerance. However, the NOX complex involved in these processes is not known. The objective of these studies is to identify a potential role for the NOX2 complex, an enzyme involved in inflammation. Mice lacking the catalytic subunit of NOX2 (Nox2?/?) or its regulatory subunit, p47phox (p47phox?/?), developed antinociceptive tolerance similar to wildtype (wt) mice after three days of continuous morphine. However, while wt mice continue to develop tolerance by day six, morphine analgesia was restored in both Nox2?/? and p47phox?/? mice. Moreover, the loss of Nox2 or p47 did not affect acute morphine analgesia in naďve mice. In wt mice, antinociceptive tolerance was associated with increased activation of NOX, nitration of MnSOD, and proinflammatory cytokines production in spinal cord. These events were markedly attenuated in Nox2?/? and p47phox?/? mice and instead, there was enhanced formation of antiinflammatory cytokine (IL4 and IL10) production. These results suggest that NOX2 activity provides a significant source of superoxide-derived PN to undertake post-translational modifications of mitochondrial MnSOD and to engage neuroinflammatory signaling in the spinal cord associated with opioid-induced antinociceptive tolerance. Thus, NOX2 may provide a potential target for adjuvant therapy to protect opioid analgesia. PMID:23454539

Doyle, Timothy; Esposito, Emanuela; Bryant, Leesa; Cuzzocrea, Salvatore; Salvemini, Daniela

2013-01-01

40

Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation  

PubMed Central

In animals and plants, pathogen recognition triggers the local activation of intracellular signaling that is prerequisite for mounting systemic defenses in the whole organism. We identified that Arabidopsis thaliana isoform CPK5 of the plant calcium-dependent protein kinase family becomes rapidly biochemically activated in response to pathogen-associated molecular pattern (PAMP) stimulation. CPK5 signaling resulted in enhanced salicylic acid–mediated resistance to the bacterial pathogen Pst DC3000, differential plant defense gene expression, and synthesis of reactive oxygen species (ROS). Using selected reaction monitoring MS, we identified the plant NADPH oxidase, respiratory burst oxidase homolog D (RBOHD), as an in vivo phosphorylation target of CPK5. Remarkably, CPK5-dependent in vivo phosphorylation of RBOHD occurs on both PAMP- and ROS stimulation. Furthermore, rapid CPK5-dependent biochemical and transcriptional activation of defense reactions at distal sites is compromised in cpk5 and rbohd mutants. Our data not only identify CPK5 as a key regulator of innate immune responses in plants but also support a model of ROS-mediated cell-to-cell communication, where a self-propagating mutual activation circuit consisting of the protein kinase, CPK5, and the NADPH oxidase RBOHD facilitates rapid signal propagation as a prerequisite for defense response activation at distal sites within the plant. PMID:23650383

Dubiella, Ullrich; Seybold, Heike; Durian, Guido; Komander, Eileen; Lassig, Roman; Witte, Claus-Peter; Schulze, Waltraud X.; Romeis, Tina

2013-01-01

41

Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation.  

PubMed

In animals and plants, pathogen recognition triggers the local activation of intracellular signaling that is prerequisite for mounting systemic defenses in the whole organism. We identified that Arabidopsis thaliana isoform CPK5 of the plant calcium-dependent protein kinase family becomes rapidly biochemically activated in response to pathogen-associated molecular pattern (PAMP) stimulation. CPK5 signaling resulted in enhanced salicylic acid-mediated resistance to the bacterial pathogen Pst DC3000, differential plant defense gene expression, and synthesis of reactive oxygen species (ROS). Using selected reaction monitoring MS, we identified the plant NADPH oxidase, respiratory burst oxidase homolog D (RBOHD), as an in vivo phosphorylation target of CPK5. Remarkably, CPK5-dependent in vivo phosphorylation of RBOHD occurs on both PAMP- and ROS stimulation. Furthermore, rapid CPK5-dependent biochemical and transcriptional activation of defense reactions at distal sites is compromised in cpk5 and rbohd mutants. Our data not only identify CPK5 as a key regulator of innate immune responses in plants but also support a model of ROS-mediated cell-to-cell communication, where a self-propagating mutual activation circuit consisting of the protein kinase, CPK5, and the NADPH oxidase RBOHD facilitates rapid signal propagation as a prerequisite for defense response activation at distal sites within the plant. PMID:23650383

Dubiella, Ullrich; Seybold, Heike; Durian, Guido; Komander, Eileen; Lassig, Roman; Witte, Claus-Peter; Schulze, Waltraud X; Romeis, Tina

2013-05-21

42

Positive feedback regulation of maize NADPH oxidase by mitogen-activated protein kinase cascade in abscisic acid signalling  

PubMed Central

In maize (Zea mays), abscisic acid (ABA)-induced H2O2 production activates a 46 kDa mitogen-activated protein kinase (p46MAPK), and the activation of p46MAPK also regulates the production of H2O2. However, the mechanism for the regulation of H2O2 production by MAPK in ABA signalling remains to be elucidated. In this study, four reactive oxygen species (ROS)-producing NADPH oxidase (rboh) genes (ZmrbohA–D) were isolated and characterized in maize leaves. ABA treatment induced a biphasic response (phase I and phase II) in the expression of ZmrbohA–D and the activity of NADPH oxidase. Phase II induced by ABA was blocked by pretreatments with two MAPK kinase (MPKKK) inhibitors and two H2O2 scavengers, but phase I was not affected by these inhibitors or scavengers. Treatment with H2O2 alone also only induced phase II, and the induction was arrested by the MAPKK inhibitors. Furthermore, the ABA-activated p46MAPK was partially purified. Using primers corresponding to the sequences of internal tryptic peptides, the p46MAPK gene was cloned. Analysis of the tryptic peptides and the p46MAPK sequence indicate it is the known ZmMPK5. Treatments with ABA and H2O2 led to a significant increase in the activity of ZmMPK5, although ABA treatment only induced a slight increase in the expression of ZmMPK5. The data indicate that H2O2-activated ZmMPK5 is involved in the activation of phase II in ABA signalling, but not in phase I. The results suggest that there is a positive feedback loop involving NADPH oxidase, H2O2, and ZmMPK5 in ABA signalling. PMID:19592501

Lin, Fan; Ding, Haidong; Wang, Jinxiang; Zhang, Hong; Zhang, Aying; Zhang, Yun; Tan, Mingpu; Dong, Wen; Jiang, Mingyi

2009-01-01

43

Antioxidant effect of imperatorin from Angelica dahurica in hypertension via inhibiting NADPH oxidase activation and MAPK pathway.  

PubMed

Imperatorin (IMP) is an active furocoumarin in the traditional Chinese medicine Angelica dahurica and has been demonstrated to have vasodilatory activity. In the present study, we investigated the effect of IMP on blood pressure (BP) and antioxidant effects in spontaneously hypertensive rats (SHR) and human embryonic kidney 293 cells. SHR were administered IMP (6.25, 12.5, and 25 mg/kg/d) or tempol (18 mg/kg/d) daily by gavage for 12 weeks. Thiobarbituric acid-reactive substances, proteinuria levels, and superoxide dismutase activity were evaluated with commercial kits. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits of the renal cortical tissues were determined by reverse transcriptase polymerase chain reaction and Western blot. Twenty-four hour urinary 8-Iso-prostaglandin F2? was measured by enzyme linked immunosorbent assay. Systolic BP and diastolic BP were significantly reduced by treatment with IMP (6.25, 12.5, and 25 mg/kg/d) in SHR. Meanwhile, we found that renal cortical superoxide dismutase activities were significantly increased in IMP-treated groups. Renal cortical and urinary thiobarbituric acid-reactive substances' levels, the 24-hour urinary excretion of 8-Iso-prostaglandin F2?, and proteinuria in the IMP-treated group, were lower than SHR group. After that, we found the messenger RNA expressions and protein levels of NADPH oxidase subunits were markedly reduced after IMP treated in SHR. IMP also reduced the phosphorylation of protein kinase B, extracellular signal-regulated kinase1/2, p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase in renal cortical in SHR. In addition, H2O2-induced ROS production in human embryonic kidney 293 cells was markedly attenuated by IMP. H2O2-induced activation of MAPK, protein kinase B, and expression of NADPH oxidase were also attenuated by pretreatment of IMP. In summary, IMP showed antihypertensive effect via prevention of renal injury not only by reducing NADPH oxidase but also by inhibiting of MAPK pathway. PMID:24973900

Cao, Yanjun; Zhang, Yanmin; Wang, Nan; He, Langchong

2014-08-01

44

NecroX-7 prevents oxidative stress-induced cardiomyopathy by inhibition of NADPH oxidase activity in rats  

SciTech Connect

Oxidative stress is one of the causes of cardiomyopathy. In the present study, NecroXs, novel class of mitochondrial ROS/RNS scavengers, were evaluated for cardioprotection in in vitro and in vivo model, and the putative mechanism of the cardioprotection of NecroX-7 was investigated by global gene expression profiling and subsequent biochemical analysis. NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced death of H9C2 rat cardiomyocytes at EC{sub 50} = 0.057 ?M. In doxorubicin (DOX)-induced cardiomyopathy in rats, NecroX-7 significantly reduced the plasma levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) which were increased by DOX treatment (p < 0.05). Microarray analysis revealed that 21 genes differentially expressed in tBHP-treated H9C2 cells were involved in ‘Production of reactive oxygen species’ (p = 0.022), and they were resolved by concurrent NecroX-7 treatment. Gene-to-gene networking also identified that NecroX-7 relieved cell death through Ncf1/p47phox and Rac2 modulation. In subsequent biochemical analysis, NecroX-7 inhibited NADPH oxidase (NOX) activity by 53.3% (p < 0.001). These findings demonstrate that NecroX-7, in part, provides substantial protection of cardiomyopathy induced by tBHP or DOX via NOX-mediated cell death. -- Highlights: ? NecroX-7 prevented tert-butyl hydroperoxide-induced in vitro cardiac cell death. ? NecroX-7 ameliorated doxorubicin-induced in vivo cardiomyopathy. ? NecroX-7 prevented oxidative stress and necrosis-enriched transcriptional changes. ? NecroX-7 effectively inhibited NADPH oxidase activation. ? Cardioprotection of Necro-7 was brought on by modulation of NADPH oxidase activity.

Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun; Kim, Hyoung Jin [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of)] [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Park, Ji-hoon [Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of)] [Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Koo, Sun Young; Kwak, Hyo-Shin; Park, Heui Sul; Kim, Dong Wook; Song, Myoungsub; Yim, Hyeon Joo; Seo, Dong Ook [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of)] [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Kim, Soon Ha, E-mail: shakim@lgls.com [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of)

2012-08-15

45

Fulvene-5 inhibition of Nadph oxidases attenuates activation of epithelial sodium channels in A6 distal nephron cells  

PubMed Central

Nadph oxidase 4 is an important cellular source of reactive oxygen species (ROS) generation in the kidney. Novel antioxidant drugs, such as Nox4 inhibitor compounds, are being developed. There is, however, very little experimental evidence for the biological role and regulation of Nadph oxidase isoforms in the kidney. Herein, we show that Fulvene-5 is an effective inhibitor of Nox-generated ROS and report the role of Nox isoforms in activating epithelial sodium channels (ENaC) in A6 distal nephron cells via oxidant signaling and cell stretch activation. Using single-channel patch-clamp analysis, we report that Fulvene-5 blocked the increase in ENaC activity that is typically observed with H2O2 treatment of A6 cells: average ENaC NPo values decreased from a baseline level of 1.04 ± 0.18 (means ± SE) to 0.25 ± 0.08 following Fulvene-5 treatment. H2O2 treatment failed to increase ENaC activity in the presence of Fulvene-5. Moreover, Fulvene-5 treatment of A6 cells blocked the osmotic cell stretch response of A6 cells, indicating that stretch activation of Nox-derived ROS plays an important role in ENaC regulation. Together, these findings indicate that Fulvene-5, and perhaps other classes of antioxidant inhibitors, may represent a novel class of compounds useful for the treatment of pathological disorders stemming from inappropriate ion channel activity, such as hypertension. PMID:23863470

Trac, David; Liu, Bingchen; Pao, Alan C.; Thomas, Sheela V.; Park, Michael; Downs, Charles A.; Ma, He-Ping

2013-01-01

46

HDAC4/5-HMGB1 signalling mediated by NADPH oxidase activity contributes to cerebral ischaemia/reperfusion injury.  

PubMed

Histone deacetylases (HDACs)-mediated epigenetic mechanisms play critical roles in the homeostasis of histone acetylation and gene transcription. HDAC inhibitors have displayed neuroprotective properties in animal models for various neurological diseases including Alzheimer's disease and ischaemic stroke. However, some studies have also reported that HDAC enzymes exert protective effects in several pathological conditions including ischaemic stress. The mixed results indicate the specific roles of each HDAC protein in different diseased states. However, the subtypes of HDACs associated with ischaemic stroke keep unclear. Therefore, in this study, we used an in vivo middle cerebral artery occlusion (MCAO) model and in vitro cell cultures by the model of oxygen glucose deprivation to investigate the expression patterns of HDACs and explore the roles of individual HDACs in ischaemic stroke. Our results showed that inhibition of NADPH oxidase activity ameliorated cerebral ischaemia/reperfusion (I/R) injury and among Zn(2+) -dependent HDACs, HDAC4 and HDAC5 were significantly decreased both in vivo and in vitro, which can be reversed by NADPH oxidase inhibitor apocynin. We further found that both HDAC4 and HDAC5 increased cell viability through inhibition of HMGB1, a central mediator of tissue damage following acute injury, expression and release in PC12 cells. Our results for the first time provide evidence that NADPH oxidase-mediated HDAC4 and HDAC5 expression contributes to cerebral ischaemia injury via HMGB1 signalling pathway, suggesting that it is important to elucidate the role of individual HDACs within the brain, and the development of HDAC inhibitors with improved specificity is required to develop effective therapeutic strategies to treat stroke. PMID:23480850

He, Min; Zhang, Bin; Wei, Xinbing; Wang, Ziying; Fan, Baoxia; Du, Pengchao; Zhang, Yan; Jian, Wencheng; Chen, Lin; Wang, Linlin; Fang, Hao; Li, Xiang; Wang, Ping-An; Yi, Fan

2013-04-01

47

New Insights into the Regulation of Neutrophil NADPH Oxidase Activity in the Phagosome: A Focus on the Role of Lipid and Ca2+ Signaling  

PubMed Central

Abstract Significance: Reactive oxygen species, produced by the phagosomal NADPH oxidase of neutrophils, play a significant physiological role during normal defense. Their role is not only to kill invading pathogens, but also to act as modulators of global physiological functions of phagosomes. Given the importance of NADPH oxidase in the immune system, its activity has to be decisively controlled by distinctive mechanisms to ensure appropriate regulation at the phagosome. Recent Advances: Here, we describe the signal transduction pathways that regulate phagosomal NADPH oxidase in neutrophils, with an emphasis on the role of lipid metabolism and intracellular Ca2+ mobilization. Critical Issues: The potential involvement of Ca2+-binding S100A8 and S100A9 proteins, known to interact with the plasma membrane NADPH oxidase, is also considered. Future Directions: Recent technical progress in advanced live imaging microscopy will permit to focus more accurately on phagosomal rather than plasma membrane NADPH oxidase regulation during neutrophil phagocytosis. Antioxid. Redox Signal. 18, 661–676. PMID:22867131

Brechard, Sabrina; Plancon, Sebastien

2013-01-01

48

Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease  

PubMed Central

Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease (SCD). One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle red blood cells (RBC). Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. In this study, we demonstrate that a significant part of ROS production in sickle cells is mediated enzymatically by NADPH oxidase, which is regulated by protein kinase C, Rac GTPase, and intracellular Ca2+ signaling within the sickle RBC. Moreover, plasma from patients with SCD and isolated cytokines, such as transforming growth factor ?1 and endothelin-1, enhance RBC NADPH oxidase activity and increase ROS generation. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte rigidity and fragility in SCD. Erythrocyte ROS generation, hemolysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive-feedback loop to drive the pathophysiology of sickle cell disease. These findings suggest a novel pathogenic mechanism in SCD and may offer new therapeutic targets to counteract inflammation and RBC rigidity and fragility in SCD. PMID:23349388

Pushkaran, Suvarnamala; Konstantinidis, Diamantis G.; Koochaki, Sebastian; Malik, Punam; Mohandas, Narla; Zheng, Yi; Joiner, Clinton H.; Kalfa, Theodosia A.

2013-01-01

49

Constitutive NADPH oxidase 4 activity resides in the composition of the B-loop and the penultimate C terminus.  

PubMed

Redox regulation of signaling molecules contributes critically to propagation of intracellular signals. The main source providing reactive oxygen species (ROS) for these physiological processes are activated NADPH oxidases (Nox/Duox family). In a pathophysiological context, some NADPH oxidase complexes produce large amounts of ROS either as part of the antimicrobial immune defense or as pathologic oxidative stress in many chronic diseases. Thus, understanding the switch from a dormant, inactive conformation to the active state of these enzymes will aid the development of inhibitors. As exogenously expressed Nox4 represents the only constitutively active enzyme in this family, analysis of structural determinants that permit this active conformation was undertaken. Our focus was directed toward a cell-based analysis of the first intracellular loop, the B-loop, and the C-terminus, two regions of Nox family enzymes that are essential for electron transfer. Mutagenesis of the B-loop identified several unique residues and a polybasic motif that contribute to the catalytic activity of Nox4. By using a multifaceted approach, including Nox4-Nox2 chimeras, mutagenesis, and insertion of Nox2 domains, we show here that the penultimate 22 amino acids of Nox4 are involved in constitutive ROS generation. The appropriate spacing of the C-terminal Nox4 sequence may cooperate with a discrete arginine-based interaction site in the B-loop, providing an intrinsically active interface that could not be disrupted by peptides derived from the Nox4 C-terminus. These results indicate that accessibility for a Nox4-specific peptide inhibitor might be difficult to achieve in vivo. PMID:22277655

von Löhneysen, Katharina; Noack, Deborah; Hayes, Patti; Friedman, Jeffrey S; Knaus, Ulla G

2012-03-16

50

Early Events Induced by the Elicitor Cryptogein in Tobacco Cells: Involvement of a Plasma Membrane NADPH Oxidase and Activation of Glycolysis and the Pentose Phosphate Pathway.  

PubMed Central

Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date. PMID:12237354

Pugin, A.; Frachisse, J. M.; Tavernier, E.; Bligny, R.; Gout, E.; Douce, R.; Guern, J.

1997-01-01

51

AMPK activation by glucagon-like peptide-1 prevents NADPH oxidase activation induced by hyperglycemia in adult cardiomyocytes.  

PubMed

Exposure of cardiomyocytes to high glucose concentrations (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase (NOX2). NOX2 activation is triggered by enhanced glucose transport through a sodium-glucose cotransporter (SGLT) but not by a stimulation of glucose metabolism. The aim of this work was to identify potential therapeutic approaches to counteract this glucotoxicity. In cultured adult rat cardiomyocytes incubated with 21 mM glucose (HG), AMP-activated protein kinase (AMPK) activation by A769662 or phenformin nearly suppressed ROS production. Interestingly, glucagon-like peptide 1 (GLP-1), a new antidiabetic drug, concomitantly induced AMPK activation and prevented the HG-mediated ROS production (maximal effect at 100 nM). ?2-AMPK, the major isoform expressed in cardiomyocytes (but not ?1-AMPK), was activated in response to GLP-1. Anti-ROS properties of AMPK activators were not related to changes in glucose uptake or glycolysis. Using in situ proximity ligation assay, we demonstrated that AMPK activation prevented the HG-induced p47phox translocation to caveolae, whatever the AMPK activators used. NOX2 activation by either ?-methyl-d-glucopyranoside, a glucose analog transported through SGLT, or angiotensin II was also counteracted by GLP-1. The crucial role of AMPK in limiting HG-mediated NOX2 activation was demonstrated by overexpressing a constitutively active form of ?2-AMPK using adenoviral infection. This overexpression prevented NOX2 activation in response to HG, whereas GLP-1 lost its protective action in ?2-AMPK-deficient mouse cardiomyocytes. Under HG, the GLP-1/AMPK pathway inhibited PKC-?2 phosphorylation, a key element mediating p47phox translocation. In conclusion, GLP-1 induces ?2-AMPK activation and blocks HG-induced p47phox translocation to the plasma membrane, thereby preventing glucotoxicity. PMID:25128166

Balteau, Magali; Van Steenbergen, Anne; Timmermans, Aurélie D; Dessy, Chantal; Behets-Wydemans, Gaetane; Tajeddine, Nicolas; Castanares-Zapatero, Diego; Gilon, Patrick; Vanoverschelde, Jean-Louis; Horman, Sandrine; Hue, Louis; Bertrand, Luc; Beauloye, Christophe

2014-10-15

52

Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function  

PubMed Central

Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates a number of functions of these organelles that allow them to participate in processes essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3-inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3-inflammasome and caspase-1 in host defense. PMID:23644505

Sokolovska, Anna; Becker, Christine E.; Eddie Ip, WK; Rathinam, Vijay A.K.; Brudner, Matthew; Paquette, Nicholas; Tanne, Antoine; Vanaja, Sivapriya K.; Moore, Kathryn J.; Fitzgerald, Katherine A.; Lacy-Hulbert, Adam; Stuart, Lynda M.

2013-01-01

53

Inhibition of arsenic induced-rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-{beta}/Smad activation  

SciTech Connect

Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30 ppm) with or without GSE (100 mg/kg, every other day by oral gavage) for 12 months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3 phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-{beta}1, type I procollagen (Coll-I) and {alpha}-smooth muscle actin ({alpha}-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-{beta}1-induced transactivation of the TGF-{beta}-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-{beta}1-induced mRNA expression of Coll-I and {alpha}-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-{beta}/Smad activation. - Research Highlights: > GSE attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. > GSE reduced arsenic-mediated Smad2/3 phosphorylation and NADPH oxidase subunits (Nox2, Nox4 and p47phox). > Beneficial effects of GSE on As-induced liver injury was via inhibition of NADPH oxidase and TGF-{beta}/Smad activation.

Pan Xinjuan; Dai Yujie; Li Xing; Niu Nannan; Li Wenjie; Liu Fangli; Zhao Yang; Yu Zengli, E-mail: zly@zzu.edu.cn

2011-08-01

54

NADPH oxidases: novel therapeutic targets for neurodegenerative diseases  

PubMed Central

Oxidative stress is a key pathologic factor in neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. The failure of free-radical-scavenging antioxidants in clinical trials pinpoints an urgent need to identify and to block major sources of oxidative stress in neurodegenerative diseases. As a major superoxide-producing enzyme complex in activated phagocytes, phagocytic NADPH oxidase (PHOX) is essential for host defense. However, recent preclinical evidence has underscored a pivotal role of over-activated PHOX in chronic neuroinflammation and progressive neurodegeneration. Deficiency in PHOX subunits mitigates neuronal damage induced by diverse insults/stresses relevant to neurodegenerative diseases. More importantly, the suppression of PHOX activity correlates with less neuronal impairment in models of neurodegenerative diseases. The discovery of PHOX and non-phagocytic NADPH oxidases in astroglia and neurons further reinforces the critical role of NADPH oxidases in oxidative stress-mediated chronic neurodegeneration. Thus, proper modulation of NADPH oxidase activity might hold therapeutic potential for currently incurable neurodegenerative diseases. PMID:22503440

Gao, Hui-Ming; Zhou, Hui; Hong, Jau-Shyong

2012-01-01

55

Human recombinant cytochrome p450 enzymes display distinct hydrogen peroxide generating activities during substrate independent NADPH oxidase reactions.  

PubMed

Microsomal enzymes generate H2O2 in the presence of NADPH. In this reaction, referred to as "oxidase" activity, H2O2 is generated directly or indirectly via the formation of superoxide anion. In the presence of redox active transition metals, H2O2 can form highly toxic hydroxyl radicals and, depending on the "oxidase" activity of individual cytochrome P450 isoenzymes, this can compromise cellular functioning and contribute to tissue injury. In the present studies, we compared the initial rates of H2O2 generating activity of microsomal preparations containing various human recombinant cytochromes P450s. In the absence of cytochrome P450s the human recombinant NADPH cytochrome P450 reductase (CPR) generated low, but detectable amounts of H2O2 (?0.04 nmol H2O2/min/100 units of reductase). Significantly greater activity was detected in preparations containing individual cytochrome P450s coexpressed with CPR (from 6.0 nmol H2O2/min/nmol P450 to 0.2 nmol/min/nmol P450); CYP1A1 was the most active, followed by CYP2D6, CYP3A4, CYP2E1, CYP4A11, CYP1A2, and CYP2C subfamily enzymes. H2O2 generating activity of the cytochrome P450s was independent of the ratio of CYP/CPR. Thus, similar H2O2 generating activity was noted with the same cytochrome P450s (CYP3A4, CYP2E1, and CYP2C9) expressed at or near the ratio of CYP/CPR in human liver microsomes (5-7), and when CPR was present in excess (CYP/CPR = 0.2-0.3). Because CYP3A4/5/7 represent up to 40% of total cytochrome P450 in the liver, these data indicate that these enzymes are the major source of H2O2 in human liver microsomes. PMID:25061110

Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

2014-10-01

56

Targeting NADPH Oxidase and Phospholipases A2 in Alzheimer's Disease  

PubMed Central

Alzheimer’s disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (A?) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by A?. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of A? in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A2 (PLA2) and secretory PLA2. In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving A? NADPH oxidase and PLA2 can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease. PMID:20195796

Simonyi, Agnes; He, Yan; Sheng, Wenwen; Sun, Albert Y.; Wood, W. Gibson; Weisman, Gary A.

2011-01-01

57

Targeting NADPH oxidase and phospholipases A2 in Alzheimer's disease.  

PubMed

Alzheimer's disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Abeta) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Abeta. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Abeta in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A(2) (PLA(2)) and secretory PLA(2). In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Abeta NADPH oxidase and PLA(2) can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease. PMID:20195796

Simonyi, Agnes; He, Yan; Sheng, Wenwen; Sun, Albert Y; Wood, W Gibson; Weisman, Gary A; Sun, Grace Y

2010-06-01

58

The mechanism of activation of NADPH oxidase in the cell-free system: the activation process is primarily catalytic and not through the formation of a stoichiometric complex.  

PubMed Central

It is commonly assumed that activation of the superoxide-generating NADPH oxidase requires the formation of a stable complex between flavocytochrome b-245 (the gp91phox/p22phox heterodimer) and the cytosolic cofactors p47phox, p67phox and Rac2. This association is thought to convert flavocytochrome b-245, which contains the NADPH-binding site, flavin and haem centres, from an inactive into an active state. Here we provide evidence that, in the cell-free system, this activation process does not necessarily require the formation of a stable stoichiometric complex between the phox proteins. To explain this data we propose the hypothesis that p67phox (and possibly Rac2), are capable of activating flavocytochrome b-245 in a catalytic fashion, where a single molecule of p67phox (or Rac2) is capable of activating multiple flavocytochrome b-245 molecules. PMID:10393079

Cross, A R; Erickson, R W; Curnutte, J T

1999-01-01

59

Neutral Sphingomyelinase Activation Precedes NADPH Oxidase-Dependent Damage in Neurons Exposed to the Proinflammatory Cytokine TNF?  

PubMed Central

Inflammation accompanied by severe oxidative stress plays a vital role in the orchestration and progression of neurodegeneration prevalent in chronic and acute CNS pathologies as well as in aging. The proinflammatory cytokine tumor necrosis factor alpha (TNF?) elicits the formation of the bioactive ceramide by stimulating the hydrolysis of the membrane lipid sphingomyelin by sphingomyelinase activities. Ceramide stimulates the formation of reactive oxygen species (ROS) and apoptotic mechanisms in both neurons and non-neuronal cells establishing a link between sphingolipid metabolism and oxidative stress. We demonstrated in SH-SY5Y human neuroblastoma cells and primary cortical neurons, that TNF? is a potent stimulator of Mg2+-dependent neutral sphingomyelinase (Mg2+-nSMase) activity and sphingomyelin hydrolysis rather than de novo synthesis, was the predominant source of ceramide increases. Mg2+-nSMase activity preceded an accumulation of ROS by a neuronal NADPH oxidase (NOX). Notably, TNF? provoked a NOX-dependent oxidative damage to sphingosine kinase-1, which generates sphingosine-1-phosphate, a ceramide metabolite associated with neurite outgrowth. Indeed, ceramide and ROS inhibited neurite outgrowth of DRG neurons by disrupting growth cone motility. Blunting ceramide and ROS formation both rescued sphingosine kinase-1 activity and neurite outgrowth. Our studies suggest that TNF?-mediated activation of Mg2+-nSMase and NOX in neuronal cells not only produced the neurotoxic intermediates ceramide and ROS but also directly antagonized neuronal survival mechanisms thus accelerating neurodegeneration. PMID:21932365

Barth, Brian M.; Gustafson, Sally J.; Kuhn, Thomas B.

2011-01-01

60

Angiotensin(1–7) Prevents Activation of NADPH Oxidase and Renal Vascular Dysfunction in Diabetic Hypertensive Rats  

Microsoft Academic Search

Background\\/Aim: We examined the influence of chronic treatment with angiotensin-(1–7) [Ang-(1–7)] on renox (renal NADPH oxidase, NOX-4) and the development of renal dysfunction in streptozotocin-treated spontaneously hypertensive rats (diabetic SHR). Methods:Mean arterial pressure, urinary protein and vascular responsiveness of the isolated renal artery to vasoactive agonists were studied in vehicle- or Ang-(1–7)-treated SHR and diabetic SHR. Results: Ang-(1–7) decreased the

Ibrahim F. Benter; Mariam H. M. Yousif; Gursev S. Dhaunsi; Jaspal Kaur; Mark C. Chappell; Debra I. Diz

2008-01-01

61

Reduction of NADPH-Oxidase Activity Ameliorates the Cardiovascular Phenotype in a Mouse Model of Williams-Beuren Syndrome  

Microsoft Academic Search

A hallmark feature of Williams-Beuren Syndrome (WBS) is a generalized arteriopathy due to elastin deficiency, presenting as stenoses of medium and large arteries and leading to hypertension and other cardiovascular complications. Deletion of a functional NCF1 gene copy has been shown to protect a proportion of WBS patients against hypertension, likely through reduced NADPH-oxidase (NOX)–mediated oxidative stress. DD mice, carrying

Victoria Campuzano; Maria Segura-Puimedon; Verena Terrado; Carolina Sánchez-Rodríguez; Mathilde Coustets; Mauricio Menacho-Márquez; Julián Nevado; Xosé R. Bustelo; Uta Francke; Luis A. Pérez-Jurado

2012-01-01

62

NADPH oxidase activity in pollen tubes is affected by calcium ions, signaling phospholipids and Rac/Rop GTPases.  

PubMed

Reactive oxygen species (ROS) generated by NADPH oxidase (NOX) are crucial for tip growth of pollen tubes. However, the regulation of NOX activity in pollen tubes remains unknown. Using purified plasma membrane fractions from tobacco and olive pollen and tobacco BY-2 cells, we demonstrate that pollen NOX is activated by calcium ions and low abundant signaling phospholipids, such as phosphatidic acid and phosphatidylinositol 4,5-bisphosphate in vitro and in vivo. Our data also suggest possible synergism between Ca(2+) and phospholipid-mediated NOX activation in pollen. Rac/Rop small GTPases are also necessary for normal pollen tube growth and have been proposed to regulate ROS production in root hairs. We show here elevated ROS formation in pollen tubes overexpressing wild-type NtRac5 and constitutively active NtRac5, while overexpression of dominant-negative NtRac5 led to a decrease of ROS in pollen tubes. We also show that PA formed by distinct phospholipases D (PLD) is involved in pathways both upstream and downstream of NOX-mediated ROS generation and identify NtPLD? as a PLD isoform acting in the ROS response pathway. PMID:22762791

Potocký, Martin; Pejchar, P?emysl; Gutkowska, Ma?gorzata; Jiménez-Quesada, María José; Potocká, Andrea; Alché, Juan de Dios; Kost, Benedikt; Žárský, Viktor

2012-11-01

63

[Influence of rhizobial (Rhizobium leguminosarum) inoculation and calcium ions on the NADPH oxidase activity in roots of etiolated pea (Pisum sativum L.) seedlings].  

PubMed

Changes in the functional activity of the NADPH oxidase in the microsomal fraction of roots of etiolated pea seedlings, caused by rhizobial inoculation and calcium ions (Ca2+), are shown. The enzyme activity in a medium with an exogenous source of Ca2+ (CaCl2, 100 microM) fluctuated, increasing 5 to 20 min and decreasing 10 and 30 min after addition. A calcium chelator (ethylene glycol tetraacetic acid (EDTA), 100 microM) potentiated the decrease in the enzyme activity in the presence of exogenous calcium. Rhizobial inoculation caused a 3.9-fold increase in the enzyme activity 5 min after inoculation compared to the control (without inoculation). The Ca(2+)-channel activator (amiodarone, 300 microM) and the Ca(2+)-channel blocker (lanthanum chloride, 400 microM) reduced the NADPH oxidase activity after rhizobial inoculation compared to the control level (without inoculation). It is concluded that Ca2+ and reactive oxygen species are involved in the regulation of the membrane NADPH oxidase activity in roots of pea seedlings. PMID:23882941

Glian'ko, A K; Ishchenko, A A

2013-01-01

64

Reduction of NADPH-oxidase activity ameliorates the cardiovascular phenotype in a mouse model of Williams-Beuren Syndrome.  

PubMed

A hallmark feature of Williams-Beuren Syndrome (WBS) is a generalized arteriopathy due to elastin deficiency, presenting as stenoses of medium and large arteries and leading to hypertension and other cardiovascular complications. Deletion of a functional NCF1 gene copy has been shown to protect a proportion of WBS patients against hypertension, likely through reduced NADPH-oxidase (NOX)-mediated oxidative stress. DD mice, carrying a 0.67 Mb heterozygous deletion including the Eln gene, presented with a generalized arteriopathy, hypertension, and cardiac hypertrophy, associated with elevated angiotensin II (angII), oxidative stress parameters, and Ncf1 expression. Genetic (by crossing with Ncf1 mutant) and/or pharmacological (with ang II type 1 receptor blocker, losartan, or NOX inhibitor apocynin) reduction of NOX activity controlled hormonal and biochemical parameters in DD mice, resulting in normalized blood pressure and improved cardiovascular histology. We provide strong evidence for implication of the redox system in the pathophysiology of the cardiovascular disease in a mouse model of WBS. The phenotype of these mice can be ameliorated by either genetic or pharmacological intervention reducing NOX activity, likely through reduced angII-mediated oxidative stress. Therefore, anti-NOX therapy merits evaluation to prevent the potentially serious cardiovascular complications of WBS, as well as in other cardiovascular disorders mediated by similar pathogenic mechanism. PMID:22319452

Campuzano, Victoria; Segura-Puimedon, Maria; Terrado, Verena; Sánchez-Rodríguez, Carolina; Coustets, Mathilde; Menacho-Márquez, Mauricio; Nevado, Julián; Bustelo, Xosé R; Francke, Uta; Pérez-Jurado, Luis A

2012-02-01

65

Dealcoholized red wine reverse vascular remodeling in an experimental model of metabolic syndrome: role of NAD(P)H oxidase and eNOS activity.  

PubMed

The present study examines the effect of chronic administration of dealcoholized red wine Malbec (DRW) on vascular remodeling and NAD(P)H oxidase and endothelial nitric oxide synthase activity (eNOS) in an experimental model of metabolic syndrome induced by fructose administration. Thirty-day old male Wistar rats were fed a normal rat diet (control) or the same diet plus 10% fructose in drinking water (FFR). During the last 4 weeks of a 10-week period of the corresponding diet, a subgroup of control and FFR (n=8 each) received DRW in their drinking water. Systolic blood pressure (SBP), a homeostasis model assessment of insulin resistance (HOMA-IR), aortic NAD(P)H oxidase and eNOS activity in the heart and vascular tissue were evaluated. Vascular remodeling was evaluated in the left carotid artery (CA) and interlobar, arcuate and interlobular renal arteries (RA) through lumen to media (L/M) ratio determination. At the end of the study FFR increased the SBP (p < 0.001), HOMA-IR (p < 0.001), and aortic NAD(P)H oxidase activity (p < 0,05) but reduced cardiac and vascular eNOS activity (p < 0.01), L/M ratio in CA (p < 0.001) and RA (p < 0.01) compared with the C group. DRW reduced SBP (p < 0.05), aortic NAD(P)H oxidase (p < 0.05), and recovered eNOS activity (p < 0.001) and L/M in CA (p < 0.001) and RA (p < 0.001) compared with FFR. This study provides new data about the beneficial effect of DRW on oxidative stress and vascular remodeling in the experimental model of metabolic syndrome. Data suggest the participation of mechanisms involving oxidative stress in FFR alterations and the usefulness of natural antioxidant substances present in red wine in the reversion of these changes. PMID:21776463

Vazquez-Prieto, Marcela Alejandra; Renna, Nicolás Federico; Lembo, Carina; Diez, Emiliano Raúl; Miatello, Roberto Miguel

2010-10-01

66

'Outside-in' signalling mechanisms underlying CD11b/CD18-mediated NADPH oxidase activation in human adherent blood eosinophils.  

PubMed

1 Incubation of human eosinophils in BSA-coated tissue culture plates resulted in time-dependent adhesion and attendant activation of the NADPH oxidase that were both inhibited (by >85%) by blocking antibodies raised against CD11b and CD18. 2 SB 203580, an inhibitor of p38 mitogen-activated protein (MAP) kinase, did not influence adhesion but inhibited superoxide anion generation (pIC50=-6.57). 3 PP1, an inhibitor of the src-family of protein tyrosine kinases, inhibited adhesion and CD11b/CD18-mediated superoxide anion generation with similar potencies (pEC50s=-5.53 and -5.99 respectively) suggesting that inhibition of the NADPH oxidase was a direct consequence of blocking adhesion. 4 The protein kinase C (PKC) inhibitors Ro-31 8220 (broad spectrum inhibitor), GF 109203X (inhibitor of conventional and novel isoforms) and Gö 6976 (inhibitor of conventional isoforms) suppressed adhesion-dependent NADPH oxidase activation (pIC50s=-6.61, -6.05 and -4.89 respectively) without affecting adhesion. Based upon the selectivity of these drugs PKCdelta and PKCepsilon are implicated in the suppression of oxidant production. 5 Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PtdIns 3-kinase), abolished superoxide anion production in adherent eosinophils (pEC50=-9.06). Similarly, CD11b/CD18-dependent adhesion was suppressed with the same potency (pEC50=-9.29) although the maximum effect did not exceed 50% implying that wortmannin also had an affect on those processes that govern adhesion-driven oxidase activation. 6 PD 098059 and piceatannol, inhibitors of MAP kinase kinase-1 and the syk tyrosine kinase respectively, had no effect on CD11b/CD18-mediated adhesion or NADPH oxidase activation. 7 The results of this study demonstrate that human eosinophils adhere to BSA-coated plastic by a CD11b/CD18-dependent mechanism, which is responsible for activation of the NADPH oxidase. Although the signalling pathway(s) utilized by CD11b/CD18 is still to be elucidated, the data presented herein implicate p38 MAP kinase, novel PKCs and PtdIns 3-kinase. PMID:10578126

Lynch, O T; Giembycz, M A; Barnes, P J; Hellewell, P G; Lindsay, M A

1999-11-01

67

Hypoxia activates NADPH oxidase to increase [ROS] i and [Ca 2+] i through the mitochondrial ROS-PKC? signaling axis in pulmonary artery smooth muscle cells  

Microsoft Academic Search

The importance of NADPH oxidase (Nox) in hypoxic responses in hypoxia-sensing cells, including pulmonary artery smooth muscle cells (PASMCs), remains uncertain. In this study, using Western blot analysis we found that the major Nox subunits Nox1, Nox4, p22phox, p47phox, and p67phox were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation

Rakesh Rathore; Yun-Min Zheng; Chun-Feng Niu; Qing-Hua Liu; Amit Korde; Ye-Shih Ho; Yong-Xiao Wang

2008-01-01

68

Activation of microglial NADPH oxidase is synergistic with glial iNOS expression in inducing neuronal death: a dual-key mechanism of inflammatory neurodegeneration  

Microsoft Academic Search

BACKGROUND: Inflammation-activated glia are seen in many CNS pathologies and may kill neurons through the release of cytotoxic mediators, such as nitric oxide from inducible NO synthase (iNOS), and possibly superoxide from NADPH oxidase (NOX). We set out to determine the relative role of these species in inducing neuronal death, and to test the dual-key hypothesis that the production of

Palwinder Mander; Guy C Brown

2005-01-01

69

Aripiprazole increases NADPH level in PC12 cells: the role of NADPH oxidase.  

PubMed

In aripiprazole-treated PC12 cells, we previously showed that the mitochondrial membrane potential (??m) was rather increased in spite of lowered cytochrome c oxidase activity. To address these inconsistent results, we focused the NADPH generation by glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway (PPP), to titrate reactive oxygen species (ROS) that results in the ??m maintenance. G6PD may be also involved in another inconsistent result of lowered intracellular lactate level in aripiprazole-treated PC12 cells, because PPP competes glucose-6-phosphate with the glycolytic pathway, resulting in the downregulation of glycolysis. Therefore, we assayed intracellular amounts of NADPH, ROS, and the activities of the enzymes generating or consuming NADPH (G6PD, NADP(+)-dependent isocitrate dehydrogenase, NADP(+)-dependent malic enzyme, glutathione reductase, and NADPH oxidase [NOX]) and estimated glycolysis in 50 ?M aripiprazole-, clozapine-, and haloperidol-treated PC12 cells. NADPH levels were enhanced only in aripiprazole-treated ones. Only haloperidol increased ROS. However, the enzyme activities did not show significant changes toward enhancing NADPH level except for the aripiprazole-induced decrease in NOX activity. Thus, the lowered NOX activity could have contributed to the aripiprazole-induced increase in the NADPH level by lowering ROS generation, resulting in maintained ??m. Although the aforementioned assumption was invalid, the ratio of fructose-1,6-bisphosphate to fructose-6-phosphate was decreased by all antipsychotics examined. Pyruvate kinase activity was enhanced only by aripiprazole. In summary, these observations indicate that aripiprazole possibly possesses the pharmacological superiority to clozapine and haloperidol in the ROS generation and the adjustment of glycolytic pathway. PMID:23934573

Nagasaki, Hiroshi; Nakashima, Akira; Kaneko, Yoko S; Kodani, Yu; Takayanagi, Takeshi; Itoh, Mitsuyasu; Kondo, Kazunao; Nagatsu, Toshiharu; Hamada, Yoji; Ota, Miyuki; Ota, Akira

2014-01-01

70

p21-Activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes.  

PubMed

Ischemic conditions reduce the activity of the p21-activated kinase (Pak1) resulting in increased arrhythmic activity. Triggered arrhythmic activity during ischemia is based on changes in cellular ionic balance and the cells Ca(2+) handling properties. In the current study we used isolated mouse ventricular myocytes (VMs) deficient for the expression of Pak1 (Pak1(-/-)) to determine the mechanism by which Pak1 influences the generation of arrhythmic activity during simulated ischemia. The Ca(2+) transient amplitude and kinetics did not significantly change in wild type (WT) and Pak1(-/-) VMs during 15 min of simulated ischemia. However, Pak1(-/-) VMs exhibited an exaggerated increase in [Ca(2+)]i, which resulted in spontaneous Ca(2+) release events and waves. The Ca(2+) overload in Pak1(-/-) VMs could be suppressed with a reverse mode blocker (KB-R7943) of the sodium calcium exchanger (NCX), a cytoplasmic scavenger of reactive oxygen species (ROS; TEMPOL) or a RAC1 inhibitor (NSC23766). Measurements of the cytoplasmic ROS levels revealed that decreased Pak1 activity in Pak1(-/-) VMs or VMs treated with the Pak1 inhibitor (IPA3) enhanced cellular ROS production. The Pak1 dependent increase in ROS was attenuated in VMs deficient for NADPH oxidase 2 (NOX2; p47(phox-/-)) or in VMs where NOX2 was inhibited (gp91ds-tat). Voltage clamp recordings showed increased NCX activity in Pak1(-/-) VMs that depended on enhanced NOX2 induced ROS production. The exaggerated Ca(2+) overload in Pak1(-/-) VMs could be mimicked by low concentrations of ouabain. Overall our data show that Pak1 is a critical negative regulator of NOX2 dependent ROS production and that a latent ROS dependent stimulation of NCX activity can predispose VMs to Ca(2+) overload under conditions where no significant changes in excitation-contraction coupling are yet evident. PMID:24380729

DeSantiago, Jaime; Bare, Dan J; Xiao, Lei; Ke, Yunbo; Solaro, R John; Banach, Kathrin

2014-02-01

71

Role of the Rho GTPase Rac in the activation of the phagocyte NADPH oxidase: outsourcing a key task.  

PubMed

The superoxide-generating NADPH oxidase of phagocytes consists of the membrane-associated cytochrome b 558 (a heterodimer of Nox2 and p22(phox)) and 4 cytosolic components: p47(phox), p67(phox), p40(phox), and the small GTPase, Rac, in complex with RhoGDI. Superoxide is produced by the NADPH-driven reduction of molecular oxygen, via a redox gradient located in Nox2. Electron flow in Nox2 is initiated by interaction with cytosolic components, which translocate to the membrane, p67(phox) playing the central role. The participation of Rac is expressed in the following sequence: (1) Translocation of the RacGDP-RhoGDI complex to the membrane; (2) Dissociation of RacGDP from RhoGDI; (3) GDP to GTP exchange on Rac, mediated by a guanine nucleotide exchange factor; (4) Binding of RacGTP to p67(phox); (5) Induction of a conformational change in p67(phox), promoting interaction with Nox2. The particular involvement of Rac in NADPH oxidase assembly serves as a paradigm for signaling by Rho GTPases, in general. PMID:24598074

Pick, Edgar

2014-01-01

72

Khz (Fusion of Ganoderma lucidum and Polyporus umbellatus Mycelia) Induces Apoptosis by Increasing Intracellular Calcium Levels and Activating JNK and NADPH Oxidase-Dependent Generation of Reactive Oxygen Species  

PubMed Central

Khz is a compound derived from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia that inhibits the growth of cancer cells. The results of the present study show that Khz induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz induced apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating JNK to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-induced apoptosis was caspase-dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the translocation of regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz. Khz triggered a rapid and sustained increase in [Ca2+]i, which activated JNK. JNK plays a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. In summary, these data indicate that Khz preferentially induces apoptosis in cancer cells, and the signaling mechanisms involve an increase in [Ca2+]i, JNK activation, and ROS generation via NADPH oxidase and mitochondria. PMID:23056263

Kim, Tae Hwan; Kim, Ju sung; Kim, Zoo haye; Huang, Ren Bin; Wang, Ren Sheng

2012-01-01

73

A Novel Nontoxic Inhibitor of the Activation of NADPH Oxidase Reduces Reactive Oxygen Species Production in Mouse LungS?  

PubMed Central

1-Hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) is a fluorinated phospholipid analog that inhibits the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6). Prdx6 PLA2 activity is required for activation of NADPH oxidase 2 and subsequent generation of reactive oxygen species (ROS). In vitro, MJ33 inhibited agonist-stimulated production of ROS by the isolated perfused mouse lung, lung microvascular endothelial cells, and polymorphonuclear leukocytes. MJ33 (0.02–0.5 µmol MJ33/kg body weight) in mixed unilamellar liposomes was administered to C57BL/6 mice by either intratracheal (i.t.) or i.v. routes. Lung MJ33 content, measured by liquid chromatography/mass spectroscopy, showed uptake of 67–87% of the injected dose for i.t. and 23–42% for i.v. administration at 4 hours postinjection. PLA2 activity of lung homogenates was markedly inhibited (>85%) at 4 hours postadministration. Both MJ33 content and PLA2 activity gradually returned to near control levels over the subsequent 24–72 hours. Mice treated with MJ33 at 12.5–25 µmol/kg did not show changes (compared with control) in clinical symptomatology, body weight, hematocrit, and histology of lung, liver, and kidney during a 30- to 50-day observation period. Thus, the toxic dose of MJ33 was >25 µmol/kg, whereas the PLA2 inhibitory dose was approximately 0.02 µmol/kg, indicating a high margin of safety. MJ33 administered to mice prior to lung isolation markedly reduced ROS production and tissue lipid and protein oxidation during ischemia followed by reperfusion. Thus, MJ33 could be useful as a therapeutic agent to prevent ROS-mediated tissue injury associated with lung inflammation or in harvested lungs prior to transplantation. PMID:23475902

Lee, Intae; Dodia, Chandra; Chatterjee, Shampa; Zagorski, John; Mesaros, Clementina; Blair, Ian A.; Feinstein, Sheldon I.; Jain, Mahendra

2013-01-01

74

Inhibition of the Human Neutrophil NADPH Oxidase by Coxiella burnetii  

PubMed Central

Coxiella burnetii is an obligate intracellular Gram-negative pathogen. A notable feature of C. burnetii is its ability to replicate within acidic phagolysosomes; however, the mechanisms utilized in evading host defenses are not well defined. Here, we investigated human neutrophil phagocytosis of C. burnetii (Nine Mile, phase II; NMII) and the effect of phagocytosed organisms on neutrophil reactive oxygen species (ROS) production. We found that opsonization with immune serum substantially enhanced phagocytosis of NMII. Human neutrophils phagocytosing opsonized NMII generated very little ROS compared to cells phagocytosing opsonized Staphylococcus aureus, Escherichia coli, or zymosan. However, phagocytosis of NMII did not affect the subsequent ROS response to a soluble agonist, indicating inhibition was localized to the phagolysosome and was not a global effect. Indeed, analysis of NADPH oxidase assembly in neutrophils after phagocytosis showed that translocation of cytosolic NADPH oxidase proteins, p47phox and p67phox, to the membrane was absent in cells phagocytosing NMII, as compared to cells phagocytosing S. aureus or activated by phorbol myristate acetate. Thus, phagocytosed NMII is able to disrupt assembly of the human neutrophil NADPH oxidase, which represents a novel virulence mechanism for this organism and appears to be a common mechanism of virulence for many intracellular pathogens. PMID:19379824

Siemsen, Daniel W.; Kirpotina, Liliya N.; Jutila, Mark A.; Quinn, Mark T.

2009-01-01

75

Characterization of the NAD(P)H Oxidase and Metronidazole Reductase Activities of the RdxA Nitroreductase of Helicobacter pylori  

PubMed Central

Metronidazole (MTZ) is widely used in combination therapies against the human gastric pathogen Helicobacter pylori. Resistance to this drug is common among clinical isolates and results from loss of function mutations in rdxA, which encodes an oxygen insensitive nitroreductase (NTR). The RdxA-associated MTZ-reductase activity of H. pylori is lost upon cell disruption. Here we provide a mechanistic explanation for this phenomenon. Under aerobic conditions, His6-tagged RdxA protein (purified from Escherichia coli), catalyzed NAD(P)H-dependent reductions of nitroaromatic and quinone substrates including: nitrofurazone, nitrofurantoin, furazolidone, CB1954, and 1,4 benzoquinone, but not MTZ. Unlike other NTRs, His6-RdxA exhibited potent NAD(P)H oxidase activity (kcat = 2.8 s?1) which suggested two possible explanations for the role of oxygen in MTZ reduction: 1) NAD(P)H oxidase activity promotes cellular hypoxia (nonspecific reduction of MTZ); and 2) molecular oxygen out-competes MTZ for reducing equivalents. The first hypothesis was eliminated upon finding that rdxA expression, while increasing MTZ toxicity in both E. coli and H. pylori constructs, did not increase paraquat toxicity even though both are of similar redox potential. The second hypothesis was confirmed by demonstrating NAD(P)H-dependent MTZ-reductase activity (apparent Km = 122 ± 58 ?M, kcat = 0.24 s?1) under strictly anaerobic conditions. The MTZ reductase activity of RdxA was 60-times greater than for NfsB (E. coli NTR), but 10 times lower than the NADPH-oxidase activity. Whether molecular oxygen directly competes with MTZ or alters the redox state of the FMN cofactors is discussed. PMID:19438716

Olekhnovich, Igor N.; Goodwin, Avery; Hoffman, Paul S.

2009-01-01

76

NADPH oxidase mediates ?-amyloid peptide-induced activation of ERK in hippocampal organotypic cultures  

Microsoft Academic Search

BACKGROUND: Previous studies have shown that beta amyloid (A?) peptide triggers the activation of several signal transduction cascades in the hippocampus, including the extracellular signal-regulated kinase (ERK) cascade. In this study we sought to characterize the cellular localization of phosphorylated, active ERK in organotypic hippocampal cultures after acute exposure to either A? (1-42) or nicotine. RESULTS: We observed that A?

Faridis Serrano; Angela Chang; Caterina Hernandez; Robia G Pautler; J David Sweatt; Eric Klann

2009-01-01

77

RXR agonists inhibit high glucose-induced upregulation of inflammation by suppressing activation of the NADPH oxidase-nuclear factor-?B pathway in human endothelial cells.  

PubMed

An inflammatory response induced by high glucose is a cause of endothelial dysfunction in diabetes and is an important contributing link to atherosclerosis. Diabetes is an independent risk factor of atherosclerosis and activation of retinoid X receptor (RXR) has been shown to exert anti-atherogenic effects. In the present study, we examined the effects of the RXR ligands 9-cis-retinoic acid (9-cis-RA) and SR11237 on high glucose-induced inflammation in human umbilical endothelial vein endothelial cells (HUVECs) and explored the potential mechanism. Our results showed that the inflammation induced by high-glucose in HUVECs was mainly mediated by the activation of nuclear factor-B (NF- ?B). High glucose-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were in comparison, significantly decreased by treatment with RXR. The effect of RXR agonists was mainly due to the inhibition of NF-?B activation. Using pharmacological inhibitors and siRNA, we confirmed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was an upstream activator of NF-?B. Furthermore, RXR agonists significantly inhibited high glucose-induced activation of NADPH oxidase and significantly decreased the production of reactive oxygen species (ROS). To explore whether the rapid inhibitory effects of RXR agonists were in fact mediated by RXR, we examined the effect of RXR downregulation by RXR siRNA. Our results showed that RXR siRNA largely abrogated the effects of RXR agonists, suggesting the requirement of RXR expression. Therefore, we have shown that RXR is involved in the regulation of NADPH oxidase- NF-?B signal pathway, as the RXR ligands antagonized the inflammatory response in HUVECs induced by high glucose. PMID:24391011

Ning, R B; Zhu, J; Chai, D J; Xu, C S; Xie, H; Lin, X Y; Zeng, J Z; Lin, J X

2013-01-01

78

Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone.  

PubMed Central

We tested the hypothesis that angiotensin II-induced hypertension is associated with an increase in vascular .O2- production, and characterized the oxidase involved in this process. Infusion of angiotensin II (0.7 mg/kg per d) increased systolic blood pressure and doubled vascular .O2- production (assessed by lucigenin chemiluminescence), predominantly from the vascular media. NE infusion (2.75 mg/kg per d) produced a similar degree of hypertension, but did not increase vascular .O2- production. Studies using various enzyme inhibitors and vascular homogenates suggested that the predominant source of .O2- activated by angiotensin II infusion is an NADH/NADPH-dependent, membrane-bound oxidase. Angiotensin II-, but not NE-, induced hypertension was associated with impaired relaxations to acetylcholine, the calcium ionophore A23187, and nitroglycerin. These relaxations were variably corrected by treatment of vessels with liposome-encapsulated superoxide dismutase. When Losartan was administered concomitantly with angiotensin II, vascular .O2- production and relaxations were normalized, demonstrating a role for the angiotensin type-1 receptor in these processes. We conclude that forms of hypertension associated with elevated circulating levels of angiotensin II may have unique vascular effects not shared by other forms of hypertension because they increase vascular smooth muscle .O2- production via NADH/NADPH oxidase activation. PMID:8621776

Rajagopalan, S; Kurz, S; Munzel, T; Tarpey, M; Freeman, B A; Griendling, K K; Harrison, D G

1996-01-01

79

Increased aortic NADPH oxidase activity in rats with genetically high angiotensin-converting enzyme levels .  

E-print Network

??In humans and rats, angiotensin I-converting enzyme activity is significantly determined by a gene polymorphism. Homozygous Brown Norway rats have higher plasma angiotensin I-converting enzyme… (more)

Jalil Milad, Jorge

2005-01-01

80

Hypohalous acid-modified human serum albumin induces neutrophil NADPH oxidase activation, degranulation, and shape change.  

PubMed

Halogenated lipids, proteins, and lipoproteins formed in reactions with myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) and hypobromous acid (HOBr) can contribute to the regulation of functional activity of cells and serve as mediators of inflammation. Human serum albumin (HSA) is the major plasma protein target of hypohalous acids. This study was performed to assess the potency of HSA modified by HOCl (HSA-Cl) and HOBr (HSA-Br) to elicit selected neutrophil responses. HSA-Cl/Br were found to induce neutrophil degranulation, generation of reactive oxygen intermediates, shape change, and actin cytoskeleton reorganization. Thus HSA-Cl/Br can initially act as a switch and then as a feeder of the "inflammatory loop" under oxidative stress. In HSA-Cl/Br-treated neutrophils, monoclonal antibodies against CD18, the ? subunit of ?2 integrins, reduced the production of superoxide anion radicals and hydrogen peroxide as well as MPO exocytosis, suggesting that CD18 contributed to neutrophil activation. HSA-Cl/Br-induced neutrophil responses were also inhibited by genistein, a broad-specificity tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, supporting the notion that activation of both tyrosine kinase and PI3K may play a role in neutrophil activation by HSA modified in MPO-dependent reactions. These results confirm the hypothesis that halogenated molecules formed in vivo via MPO-dependent reactions can be considered as a new class of biologically active substances potentially able to contribute to activation of myeloid cells in sites of inflammation and serve as inflammatory response modulators. PMID:24384524

Gorudko, Irina V; Grigorieva, Daria V; Shamova, Ekaterina V; Kostevich, Valeria A; Sokolov, Alexey V; Mikhalchik, Elena V; Cherenkevich, Sergey N; Arnhold, Jürgen; Panasenko, Oleg M

2014-03-01

81

Hypoxia/oxidative stress alters the pharmacokinetics of CPU86017-RS through mitochondrial dysfunction and NADPH oxidase activation  

PubMed Central

Aim: Hypoxia/oxidative stress can alter the pharmacokinetics (PK) of CPU86017-RS, a novel antiarrhythmic agent. The aim of this study was to investigate the mechanisms underlying the alteration of PK of CPU86017-RS by hypoxia/oxidative stress. Methods: Male SD rats exposed to normal or intermittent hypoxia (10% O2) were administered CPU86017-RS (20, 40 or 80 mg/kg, ig) for 8 consecutive days. The PK parameters of CPU86017-RS were examined on d 8. In a separate set of experiments, female SD rats were injected with isoproterenol (ISO) for 5 consecutive days to induce a stress-related status, then CPU86017-RS (80 mg/kg, ig) was administered, and the tissue distributions were examined. The levels of Mn-SOD (manganese containing superoxide dismutase), endoplasmic reticulum (ER) stress sensor proteins (ATF-6, activating transcription factor 6 and PERK, PRK-like ER kinase) and activation of NADPH oxidase (NOX) were detected with Western blotting. Rat liver microsomes were incubated under N2 for in vitro study. Results: The Cmax, t1/2, MRT (mean residence time) and AUC (area under the curve) of CPU86017-RS were significantly increased in the hypoxic rats receiving the 3 different doses of CPU86017-RS. The hypoxia-induced alteration of PK was associated with significantly reduced Mn-SOD level, and increased ATF-6, PERK and NOX levels. In ISO-treated rats, the distributions of CPU86017-RS in plasma, heart, kidney, and liver were markedly increased, and NOX levels in heart, kidney, and liver were significantly upregulated. Co-administration of the NOX blocker apocynin eliminated the abnormalities in the PK and tissue distributions of CPU86017-RS induced by hypoxia/oxidative stress. The metabolism of CPU86017-RS in the N2-treated liver microsomes was significantly reduced, addition of N-acetylcysteine (NAC), but not vitamin C, effectively reversed this change. Conclusion: The altered PK and metabolism of CPU86017-RS induced by hypoxia/oxidative stress are produced by mitochondrial abnormalities, NOX activation and ER stress; these abnormalities are significantly alleviated by apocynin or NAC. PMID:24122013

Gao, Jie; Ding, Xuan-sheng; Zhang, Yu-mao; Dai, De-zai; Liu, Mei; Zhang, Can; Dai, Yin

2013-01-01

82

Inhibition of prostacyclin release by cigarette smoke extract in endothelial cells is not related to enhanced superoxide generation and NADPH-oxidase activation.  

PubMed

Exposure of human umbilical endothelial cells (ECs) to cigarette smoke extract (CSE) activated the NADPH-oxidase enzyme and increased the production of superoxide (O-2) as well as reactive oxygen species (ROS). CSE also inhibited the prostacyclin (PGI2) formation by ECs. Preincubation of ECs with diphenylene iodonium (DPI), the inhibitor of NADPH oxidase, blocked the increase of O-2 production, but neither lowered the ROS level nor prevented the inhibition of PGI2 formation in CSE-treated cells. Preincubation of ECs with a medium supplemented with 1 mM vitamin C did not decrease, but rather increased the O-2 production in CSE-treated cells. However, adding 1 mM glutathione (GSH) to vitamin C decreased the O-2 production, indicating that vitamin C was overwhelmed by the prooxidant in CS, and GSH enhanced the recycling process and spared vitamin C. The ROS level remained high in CSE-treated cells even after preincubation with vitamin C or vitamin C + GSH compared to the control cells. These results are discussed in light of the possible decrease of antioxidant enzyme activities in CSE-treated cells and the increase of cellular hydrogen peroxide (H2O2) generated from the CSE, which cause an imbalance between oxidizing species and the antioxidants producing oxidative stress in CSE-treated cells. These results demonstrate that CSE has a direct inhibitory effect on PGI2 formation and enhances the level of ROS in CSE-treated ECs, regardless of the activation of NADPH-oxidase. PMID:17073561

Mahfouz, M; Qi, Zhou; Kummerow, F A

2006-01-01

83

Niacin restriction upregulates NADPH oxidase and ROS in human keratinocytes  

PubMed Central

NAD+ is a substrate for many enzymes, including poly(ADP-ribose) polymerases and sirtuins, which are involved in fundamental cellular processes including DNA repair, stress responses, signaling, transcription, apoptosis, metabolism, differentiation, chromatin structure, and life span. Because these molecular processes are important early in cancer development, we developed a model to identify critical NAD-dependent pathways potentially important in early skin carcinogenesis. Removal of niacin from the cell culture medium allowed control of intracellular NAD. Unlike many non-immortalized human cells, HaCaT keratinocytes, which are immortalized and have a mutant p53 and aberrant NF-kB activity, become severely NAD depleted but divide indefinitely under these conditions. Niacin deficient HaCaTs develop a decreased growth rate due to an increase in apoptotic cells and an arrest in the G2/M phase of the cell cycle. Long- term survival mechanisms in niacin deficient HaCats involve accumulation of reactive oxygen species and increased DNA damage. These alterations result, at least in part, from increased expression and activity of NADPH oxidase, whose downstream effects can be reversed by nicotinamide or NADPH oxidase inhibitors. Our data support the hypothesis that glutamine is a likely alternative energy source during niacin deficiency and we suggest a model for NADPH generation important in ROS production. PMID:17997992

Benavente, Claudia A.; Jacobson, Elaine L.

2008-01-01

84

Carbon ion beams induce hepatoma cell death by NADPH oxidase-mediated mitochondrial damage.  

PubMed

Mitochondria are a major source of reactive oxygen species (ROS) and are also the target of cellular ROS. ROS damage to mitochondria leads to dysfunction that further enhances the production of mitochondrial ROS. This feed-forward vicious cycle between mitochondria and ROS induces cell death. Within a few minutes of radiation exposure, NADPH oxidase is activated to elevate the ROS level. Activated NADPH oxidase might induce the feed-forward cycle of mitochondria and this is a possible mechanism for cancer cell death induced by heavy ion irradiation. We found that after 4?Gy of (12) C(6+) ion radiation of HepG2 cells, the NADPH oxidase membrane subunit gp91(phox) was not involved in enzyme activation through increased expression; however, the subunit p47(phox) was involved in activation by being translocated to the membrane. (12) C(6+) ion radiation clearly decreased the ??m of HepG2 cells, increasing mitochondrial DNA damage and inducing cell death. Pretreatment with apocynin (APO, an NADPH oxidase inhibitor) effectively prevented the ??m decrease, mitochondrial DNA damage, and cell death induced by radiation. However, these protective effects were not observed with APO treatment after irradiation exposure. These data demonstrated that NADPH oxidase activation was an initiator in mitochondrial damage. Once mitochondria entered the feed-forward cycle, cell fate was no longer controlled by NADPH oxidase. Only antioxidants that targeted mitochondria such as MitoQ could break the cycle and release cells from death. PMID:23804302

Sun, Chao; Wang, Zhenhua; Liu, Yang; Liu, Yuanyuan; Li, Hongyan; Di, Cuixia; Wu, Zhenhua; Gan, Lu; Zhang, Hong

2014-01-01

85

NADPH oxidase and Nrf2 regulate gastric aspiration-induced inflammation and acute lung injury  

PubMed Central

Recruitment of neutrophils and release of reactive oxygen species are considered to be major pathogenic components driving acute lung injury (ALI). However, NADPH oxidase, the major source of reactive oxygen species in activated phagocytes, can paradoxically limit inflammation and injury. We hypothesized that NADPH oxidase protects against ALI by limiting neutrophilic inflammation and by activating Nrf2, a transcriptional factor that induces anti-oxidative and cytoprotective pathways. Our objective was to delineate the roles of NADPH oxidase and Nrf2 in modulating acute lung inflammation and injury in clinically relevant models of acute gastric aspiration injury, a major cause of ALI. Acid aspiration caused increased ALI (as assessed by bronchoalveolar lavage fluid albumin concentration) in both NADPH oxidase-deficient (p47phox?/?) mice and in Nrf2?/? mice compared to wild-type mice. NADPH oxidase reduced airway neutrophil accumulation, but Nrf2 decreased ALI without affecting neutrophil recovery. Acid injury resulted in a 120-fold increase in mitochondrial DNA, a pro-inflammatory and injurious product of cellular necrosis, in cell-free bronchoalveolar lavage fluid. Pharmacologic activation of Nrf2 by the triterpenoid, CDDO-Im, limited aspiration-induced ALI in wild-type mice and reduced endothelial cell injury caused by mitochondrial extract-primed human neutrophils, leading to the conclusion that NADPH oxidase and Nrf2 have coordinated, but distinct, functions in modulating inflammation and injury. These results also point to Nrf2 as a therapeutic target to limit ALI by attenuating neutrophil-induced cellular injury. PMID:23296708

Davidson, Bruce A.; Vethanayagam, R. Robert; Grimm, Melissa J.; Mullan, Barbara A.; Raghavendran, Krishnan; Blackwell, Timothy S.; Freeman, Michael L.; Ayyasamy, Vanniarajan; Singh, Keshav K.; Sporn, Michael B.; Itagaki, Kiyoshi; Hauser, Carl J.; Knight, Paul R.; Segal, Brahm H.

2012-01-01

86

Involvement of phospholipase D and NADPH-oxidase in salicylic acid signaling cascade.  

PubMed

Salicylic acid is associated with the primary defense responses to biotic stress and formation of systemic acquired resistance. However, molecular mechanisms of early cell reactions to phytohormone application are currently undisclosed. The present study investigates the participation of phospholipase D and NADPH-oxidase in salicylic acid signal transduction cascade. The activation of lipid signaling enzymes within 15 min of salicylic acid application was shown in Arabidopsis thaliana plants by measuring the phosphatidic acid accumulation. Adding of primary alcohol (1-butanol) to the incubation medium led to phosphatidylbutanol accumulation as a result of phospholipase D (PLD) action in wild-type and NADPH-oxidase RbohD deficient plants. Salicylic acid induced rapid increase in NADPH-oxidase activity in histochemical assay with nitroblue tetrazolium but the reaction was not observed in presence of 1-butanol and NADPH-oxidase inhibitor diphenylene iodide (DPI). The further physiological effect of salicylic acid and inhibitory analysis of the signaling cascade were made in the guard cell model. Stomatal closure induced by salicylic acid was inhibited by 1-butanol and DPI treatment. rbohD transgenic plants showed impaired stomatal reaction upon phytohormone effect, while the reaction to H2O2 did not differ from that of wild-type plants. Thus a key role of NADPH-oxidase D-isoform in the process of stomatal closure in response to salicylic acid has been postulated. It has enabled to predict a cascade implication of PLD and NADPH oxidase to salicylic acid signaling pathway. PMID:23500715

Kalachova, Tetiana; Iakovenko, Oksana; Kretinin, Sergii; Kravets, Volodymyr

2013-05-01

87

NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury  

PubMed Central

Multiple sclerosis is a chronic inflammatory disease of the central nervous system, associated with demyelination and neurodegeneration. The mechanisms of tissue injury are poorly understood, but recent data suggest that mitochondrial injury may play an important role in this process. Mitochondrial injury can be triggered by reactive oxygen and nitric oxide species, and we recently provided evidence for oxidative damage of oligodendrocytes and dystrophic axons in early stages of active multiple sclerosis lesions. In this study, we identified potential sources of reactive oxygen and nitrogen species through gene expression in carefully staged and dissected lesion areas and by immunohistochemical analysis of protein expression. Genome-wide microarrays confirmed mitochondrial injury in active multiple sclerosis lesions, which may serve as an important source of reactive oxygen species. In addition, we found differences in the gene expression levels of various nicotinamide adenine dinucleotide phosphate oxidase subunits between initial multiple sclerosis lesions and control white matter. These results were confirmed at the protein level by means of immunohistochemistry, showing upregulation of the subunits gp91phox, p22phox, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 in activated microglia in classical active as well as slowly expanding lesions. The subunits gp91phox and p22phox were constitutively expressed in microglia and were upregulated in the initial lesion. In contrast, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 expression were more restricted to the zone of initial damage or to lesions from patients with acute or early relapsing/remitting multiple sclerosis. Double labelling showed co-expression of the nicotinamide adenine dinucleotide phosphate oxidase subunits in activated microglia and infiltrated macrophages, suggesting the assembly of functional complexes. Our data suggest that the inflammation-associated oxidative burst in activated microglia and macrophages plays an important role in demyelination and free radical-mediated tissue injury in the pathogenesis of multiple sclerosis. PMID:22366799

Fischer, Marie T.; Sharma, Rakhi; Lim, Jamie L.; Haider, Lukas; Frischer, Josa M.; Drexhage, Joost; Mahad, Don; Bradl, Monika; van Horssen, Jack

2012-01-01

88

Peroxiredoxin 6 Phosphorylation and Subsequent Phospholipase A2 Activity Are Required for Agonist-mediated Activation of NADPH Oxidase in Mouse Pulmonary Microvascular Endothelium and Alveolar Macrophages*  

PubMed Central

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA2) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA2 activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47phox, cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA2 activity, blocked agonist-induced PLA2 activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA2s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA2 active site (S32A, H26A, and D140A), “rescued” Ang II-induced PLA2 activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA2 activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA2 activity facilitates assembly of the NOX2 complex and activation of the oxidase. PMID:21262967

Chatterjee, Shampa; Feinstein, Sheldon I.; Dodia, Chandra; Sorokina, Elena; Lien, Yu-Chin; Nguyen, Su; Debolt, Kris; Speicher, David; Fisher, Aron B.

2011-01-01

89

Peroxiredoxin 6 phosphorylation and subsequent phospholipase A2 activity are required for agonist-mediated activation of NADPH oxidase in mouse pulmonary microvascular endothelium and alveolar macrophages.  

PubMed

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA(2)) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA(2) activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47(phox), cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA(2) activity, blocked agonist-induced PLA(2) activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA(2)s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA(2) active site (S32A, H26A, and D140A), "rescued" Ang II-induced PLA(2) activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA(2) activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA(2) activity facilitates assembly of the NOX2 complex and activation of the oxidase. PMID:21262967

Chatterjee, Shampa; Feinstein, Sheldon I; Dodia, Chandra; Sorokina, Elena; Lien, Yu-Chin; Nguyen, Su; Debolt, Kris; Speicher, David; Fisher, Aron B

2011-04-01

90

Inhaled birch pollen extract induces airway hyperresponsiveness via oxidative stress but independently of pollen-intrinsic NADPH oxidase activity, or the Toll-like receptor 4-TRIF pathway  

PubMed Central

Oxidative stress in allergic asthma may result from oxidase activity or pro-inflammatory molecules in pollens. Signaling via TLR4 and its adaptor TRIF has been implicated in reactive oxygen species (ROS)-mediated acute lung injury and in T helper 2 immune responses. We investigated the contributions of oxidative stress and TLR4/TRIF signaling to experimental asthma induced by birch pollen exposure exclusively via the airways. Mice were exposed to native or heat-inactivated white birch pollen extract (BPEx) intratracheally and injected with the antioxidants, N-acetyl-L-cysteine (NAC) or dimethylthiourea (DMTU) prior to sensitization, challenge, or all allergen exposures, to assess the role of oxidative stress and pollen-intrinsic NADPH oxidase activity in allergic sensitization, inflammation and airway hyperresponsiveness (AHR). Additionally, TLR4 signaling was antagonized concomitantly with allergen exposure, or the development of allergic airway disease was evaluated in TLR4 or TRIF knockout mice. NAC inhibited BPEx-induced eosinophilic airway inflammation and AHR except when given exclusively during sensitization, whereas DMTU was inhibitory even when administered with the sensitization alone. Heat-inactivation of BPEx had no effect on the development of allergic airway disease. Oxidative stress-mediated AHR was also TLR4- and TRIF-independent, however, TLR4 deficiency decreased, while TRIF deficiency increased BPEx-induced airway inflammation. In conclusion, oxidative stress plays a significant role in allergic sensitization to pollen via the airway mucosa, but the pollen-intrinsic NADPH oxidase activity and TLR4 or TRIF signaling are unnecessary for the induction of allergic airway disease and AHR. Pollen extract does, however, activate TLR4, thereby enhancing airway inflammation which is restrained by the TRIF-dependent pathway. PMID:23776177

Shalaby, Karim H.; Allard-Coutu, Alexandra; O’Sullivan, Michael; Nakada, Emily; Qureshi, Salman T.; Day, Brian J.; Martin, James G.

2013-01-01

91

5-aminoimidazole-4-carboxamide Riboside Induces Apoptosis Through AMP-activated Protein Kinase-independent and NADPH Oxidase-dependent Pathways  

PubMed Central

It is debatable whether AMP-activated protein kinase (AMPK) activation is involved in anti-apoptotic or pro-apoptotic signaling. AICAR treatment increases AMPK-?1 phosphorylation, decreases intracellular reactive oxygen species (ROS) levels, and significantly increases Annexin V-positive cells, DNA laddering, and caspase activity in human myeloid cell. AMPK activation is therefore implicated in apoptosis. However, AMPK-?1-knockdown THP-1 cells are more sensitive to apoptosis than control THP-1 cells are, suggesting that the apoptosis is AMPK-independent. Low doses of AICAR induce cell proliferation, whereas high doses of AICAR suppress cell proliferation. Moreover, these effects are significantly correlated with the downregulation of intracellular ROS, strongly suggesting that AICAR-induced apoptosis is critically associated with the inhibition of NADPH oxidase by AICAR. Collectively, our results demonstrate that in AICAR-induced apoptosis, intracellular ROS levels are far more relevant than AMPK activation.

Wi, Sae Mi

2014-01-01

92

Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of Brassica napus leaf protoplasts.  

PubMed

Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H(2)O(2) and enzymes involved in H(2)O(2) generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H(2)O(2) and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H(2)O(2) was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H(2)O(2) in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H(2)O(2) generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential. PMID:21853253

Tewari, Rajesh Kumar; Watanabe, Daisuke; Watanabe, Masami

2012-01-01

93

ATL9, a RING Zinc Finger Protein with E3 Ubiquitin Ligase Activity Implicated in Chitin- and NADPH Oxidase-Mediated Defense Responses  

PubMed Central

Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 (?=?ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst. PMID:21203445

Berrocal-Lobo, Marta; Stone, Sophia; Yang, Xin; Antico, Jay; Callis, Judy; Ramonell, Katrina M.; Somerville, Shauna

2010-01-01

94

Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: role of NADPH oxidase.  

PubMed

The endocannabinoid system (CS) has been implicated in the development of hepatic fibrosis such as schistosomiasis-associated liver fibrosis (SSLF). However, the mechanisms mediating the action of the CS in hepatic fibrosis are unclear. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor 1 (CB1) due to activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). The SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin, and HSCs from control and infected mice were then isolated, cultured, and confirmed by analysis of HSC markers ?-SMA and desmin. CB1 significantly increased in HSCs isolated from mice with SSLF, which was accompanied by a greater expression of fibrotic markers ?-SMA, collagen I, and TIMP-1. CB1 upregulation and enhanced fibrotic changes were also observed in normal HSCs treated with soluble egg antigen (SEA) from Schistosoma J. Electron spin resonance (ESR) analysis further demonstrated that superoxide (O2(-)) production was increased in infected HSCs or normal HSCs stimulated with SEA. Both Nox4 and Nox1 siRNA prevented SEA-induced upregulation of CB1, ?-SMA, collagen I, and TIMP-1 by inhibition of O2(-) production, while CB1 siRNA blocked SEA-induced fibrotic changes without effect on O2(-) production in these HSCs. Taken together, these data suggest that the fibrotic activation of HSCs on Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase-mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF. PMID:24657416

Wang, Mi; Abais, Justine M; Meng, Nan; Zhang, Yang; Ritter, Joseph K; Li, Pin-Lan; Tang, Wang-Xian

2014-06-01

95

Tumor necrosis factor alpha activates transcription of the NADPH oxidase organizer 1 (NOXO1) gene and upregulates superoxide production in colon epithelial cells.  

PubMed

NADPH oxidase 1 (Nox1) is a multicomponent enzyme consisting of p22(phox), Nox organizer 1 (NOXO1), Nox1 activator 1, and Rac1. Interleukin-1beta, flagellin, interferon-gamma, and tumor necrosis factor alpha (TNF-alpha) similarly induced Nox1 in a colon cancer cell line (T84), whereas only TNF-alpha fully induced NOXO1 and upregulated superoxide-producing activity by ninefold. This upregulation was canceled by knockdown of NOXO1 with small interfering RNAs. TNF-alpha rapidly phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase 1/2, followed by phosphorylation of c-Jun and c-Fos and appearance of an AP-1 binding activity within 30 min. We cloned the 5' flank of the human NOXO1 gene (-3888 to +263 bp), and found that the region between -585 and -452 bp, which contains consensus elements of YY-1, AP-1, and Ets, and the GC-rich region encoding three putative binding sites for SP-1, was crucial for TNF-alpha-dependent promoter activity. Serial mutation analysis of the elements identified an AP-1 binding site (from -561 to -551 bp, agtAAGtcatg) as a crucial element for TNF-alpha-stimulated transcription of the human NOXO1 gene, which was also confirmed by the AP-1 decoy experiments. Thus, TNF-alpha acts as a potent activator of Nox1-based oxidase in colon epithelial cells, suggesting a potential role of this oxidase in inflammation of the colon. PMID:18929641

Kuwano, Yuki; Tominaga, Kumiko; Kawahara, Tsukasa; Sasaki, Hidekazu; Takeo, Keiko; Nishida, Kensei; Masuda, Kiyoshi; Kawai, Tomoko; Teshima-Kondo, Shigetada; Rokutan, Kazuhito

2008-12-15

96

Globular adiponectin inhibits ethanol-induced reactive oxygen species production through modulation of NADPH oxidase in macrophages: involvement of liver kinase B1/AMP-activated protein kinase pathway.  

PubMed

Adiponectin, an adipokine predominantly secreted from adipocytes, has been shown to play protective roles against chronic alcohol consumption. Although excessive reactive oxygen species (ROS) production in macrophages is considered one of the critical events for ethanol-induced damage in various target tissues, the effect of adiponectin on ethanol-induced ROS production is not clearly understood. In the present study, we investigated the effect of globular adiponectin (gAcrp) on ethanol-induced ROS production and the potential mechanisms underlying these effects of gAcrp in macrophages. Here we demonstrated that gAcrp prevented ethanol-induced ROS production in both RAW 264.7 macrophages and primary murine peritoneal macrophages. Globular adiponectin also inhibited ethanol-induced activation of NADPH oxidase. In addition, gAcrp suppressed ethanol-induced increase in the expression of NADPH oxidase subunits, including Nox2 and p22(phox), via modulation of nuclear factor-?B pathway. Furthermore, pretreatment with compound C, a selective inhibitor of AMPK, or knockdown of AMPK by small interfering RNA restored suppression of ethanol-induced ROS production and Nox2 expression by gAcrp. Finally, we found that gAcrp treatment induced phosphorylation of liver kinase B1 (LKB1), an upstream signaling molecule mediating AMPK activation. Knockdown of LKB1 restored gAcrp-suppressed Nox2 expression, suggesting that LKB1/AMPK pathway plays a critical role in the suppression of ethanol-induced ROS production and activation of NADPH oxidase by gAcrp. Taken together, these results demonstrate that globular adiponectin prevents ethanol-induced ROS production, at least in part, via modulation of NADPH oxidase in macrophages. Further, LKB1/AMPK axis plays an important role in the suppression of ethanol-induced NADPH oxidase activation by gAcrp in macrophages. PMID:24850909

Kim, Mi Jin; Nagy, Laura E; Park, Pil-Hoon

2014-09-01

97

Early signalling events implicated in leukotriene B4-induced activation of the NADPH oxidase in eosinophils: role of Ca2+, protein kinase C and phospholipases C and D.  

PubMed Central

The early signalling events that may ultimately contribute to the assembly and subsequent activation of the NADPH oxidase in guinea-pig peritoneal eosinophils were investigated in response to leukotriene B4 (LTB4). LTB4 promoted a rapid, transient and receptor-mediated increase in the rate of H2O2 generation that was potentiated by R 59 022, a diradylglycerol (DRG) kinase inhibitor, implicating protein kinase C (PKC) in the genesis of this response. This conclusion was supported by the finding that the PKC inhibitor, Ro 31-8220, attenuated (by about 30%) the peak rate of LTB4-induced H2O2 generation under conditions where the same response evoked by 4 beta-phorbol 12,13-dibutyrate (PDBu) was inhibited by more than 90%. Paradoxically, Ro 31-8220 doubled the amount of H2O2 produced by LTB4 which may relate to the ability of PKC to inhibit cell signalling through phospholipase C (PLC). Indeed, Ro 31-8220 significantly enhanced LTB4-induced Ins(1,4,5)P3 accumulation and the duration of the Ca2+ transient in eosinophils. Experiments designed to assess the relative importance of DRG-mobilizing phospholipases in LTB4-induced oxidase activation indicated that phospholipase D (PLD) did not play a major role. Thus, although H2O2 generation was abolished by butan-1-ol, this was apparently unrelated to the inhibition of PLD, as LTB4 failed to stimulate the formation of Ptd[3H]BuOH in [3H]butan-1-ol-treated eosinophils. Rather, the inhibition was probably due to the ability of butan-1-ol to increase the eosinophil cyclic AMP content. In contrast, Ca(2+)- and PLC-driven mechanisms were implicated in H2O2 generation, as LTB4 elevated the Ins(1,4,5)P3 content and intracellular free Ca2+ concentration in intact cells, and cochelation of extracellular and intracellular Ca2+ significantly attenuated LTB4-induced H2O2 generation. Pretreatment of eosinophils with wortmannin did not affect LTB4-induced H2O2 production at concentrations at which it abolished the respiratory burst evoked by formylmethionyl-leucylphenylalanine in human neutrophils. Collectively, these data suggest that LTB4 activates the NADPH oxidase in eosinophils by PLD- and PtdIns 3-kinase-independent mechanisms that involve Ca2+, PLC and PKC. Furthermore, the activation of additional pathways that do not require Ca2+ is also suggested by the finding that LTB4 evoked a significant respiratory burst in Ca(2+)-depleted cells. PMID:7575412

Perkins, R S; Lindsay, M A; Barnes, P J; Giembycz, M A

1995-01-01

98

Thapsigargin Triggers Cardiac Contractile Dysfunction via NADPH Oxidase -Mediated Mitochondrial Dysfunction: Role of Akt Dephosphorylation  

PubMed Central

ER stress triggers myocardial contractile dysfunction although the underlying mechanism is still elusive. Given that NADPH oxidase was recently implicated in ER stress-induced tissue injury, this study was designed to examine the role of NADPH oxidase in the ER stress-induced cardiac mechanical defects and the impact of Akt activation on ER stress-induced cardiac anomalies. WT and transgenic mice with cardiac-specific overexpression of active mutant of Akt (MyAkt) were subjected to the ER stress inducer thapsigargin (1 and 3 mg/kg, i.p. for 48 hr). Thapsigargin compromised echocardiographic parameters including elevated LVESD and reduced fractional shortening, suppressed cardiomyocyte contractile function, intracellular Ca2+ handling and cell survival, along with enhanced carbonyl formation, apoptosis, superoxide production, NADPH oxidase expression and mitochondrial damage. Interestingly, these anomalies were attenuated or mitigated by chronic Akt activation. Treatment with thapsigargin also dephosphorylated Akt and its downstream signal GSK3? (leading to activation of GSK3?), the effect of which was abrogated in MyAkt hearts. Knockdown of cytosolic subunit of NADPH oxidase p47phox using siRNA abrogated thapsigargin-induced apoptosis and cell death in H9C2 myoblasts. In vitro exposure of thapsigargin induced murine cardiomyocyte dysfunction reminiscent of the in vivo setting, the effects of which were ablated by the NADPH oxidase inhibitor apocynin and the mitochondrial Ca2+ uptake inhibitor Ru360. In addition, apocynin abrogated thapsigargin-induced loss of mitochondrial membrane potential and permeation pore opening, similar to those induced by chronic Akt activation. In summary, these data suggest that ER stress interrupts cardiac contractile and intracellular Ca2+ homeostasis, cell survival and mitochondrial integrity through an Akt dephosphorylation and NADPH oxidase-dependent mechanism. PMID:21996563

Zhang, Yingmei; Ren, Jun

2011-01-01

99

Properties of phagocyte NADPH oxidase p47-phox mutants with unmasked SH3 (Src homology 3) domains: full reconstitution of oxidase activity in a semi-recombinant cell-free system lacking arachidonic acid.  

PubMed Central

In an early step in the assembly of the phagocyte NADPH oxidase, p47-phox translocates from the cytosol to the membrane, mediated by engagement of the N-termini of two p47-phox Src homology 3 (SH3) domains with a proline-rich region (PRR) in the p22-phox subunit of cytochrome b (558). In response to phagocyte activation, several serine residues in a C-terminal arginine/lysine-rich domain of p47-phox are phosphorylated, leading to changes in the conformation of p47-phox and exposure of its N-terminal SH3 domain that is normally masked by internal association with the arginine/lysine-rich domain. We report that triple alanine substitutions at Asp-217, Glu-218 and Glu-223 in a short sequence that links the tandem p47-phox SH3 domains unmasked the N-terminal SH3 domain, similar to the effects of aspartic acid substitutions at Ser-310 and Ser-328 in the arginine/lysine-rich region. Recombinant p47-phox proteins with mutations in either the linker region or the arginine/lysine-rich domain were active in the absence of arachidonic acid stimulation in a cell-free NADPH oxidase system consisting of recombinant p67-phox, Rac1-guanosine 5'-[gamma-thio]triphosphate and neutrophil membranes. Supplementing neutrophil membranes with phosphoinositides or other negatively charged phospholipids markedly enhanced cell-free superoxide generation by these p47-phox mutants in the absence of arachidonic acid, to levels equivalent to those generated by wild-type p47-phox following arachidonic acid activation. This enhancement may be related to recruitment to the membrane of p47-phox mediated by a novel secondary phox homology (PX) domain binding site that broadly recognizes phospholipids. No specific enhancement by specific phosphorylated phosphatidylinositols was found to suggest a dominant role for the p47-phox primary PX domain binding site. Truncated p47-phox S310D S328D lacking the C-terminal PRR was inactive in the cell-free system without arachidonic acid, but was fully active with arachidonic acid. This suggests that activation of NADPH oxidase in an arachidonate-free cell-free system requires association of the p47-phox C-terminal PRR with the p67-phox C-terminal SH3 domain. PMID:12650641

Peng, Guihong; Huang, Jin; Boyd, Mellonie; Kleinberg, Michael E

2003-01-01

100

Mechanisms for suppressing NADPH oxidase in the vascular wall  

Microsoft Academic Search

Oxidative stress underlies many forms of vascular disease as well as tissue injury following ischemia and reperfusion. The major source of oxidative stress in the artery wall is an NADPH oxidase. This enzyme complex as expressed in vascular cells differs from that in phagocytic leucocytes both in biochemical structure and functions. The crucial flavin-containing catalytic subunits, Nox1 and Nox4, are

Gregory J Dusting; Stavros Selemidis; Fan Jiang

2005-01-01

101

Calcineurin A? regulates NADPH oxidase (Nox) expression and activity via nuclear factor of activated T cells (NFAT) in response to high glucose.  

PubMed

Hypertrophy is an adaptive response that enables organs to appropriately meet increased functional demands. Previously, we reported that calcineurin (Cn) is required for glomerular and whole kidney hypertrophy in diabetic rodents (Gooch, J. L., Barnes, J. L., Garcia, S., and Abboud, H. E. (2003). Calcineurin is activated in diabetes and is required for glomerular hypertrophy and ECM accumulation. Am. J. Physiol. Renal Physiol. 284, F144-F154; Reddy, R. N., Knotts, T. L., Roberts, B. R., Molkentin, J. D., Price, S. R., and Gooch, J. L. (2011). Calcineurin A? is required for hypertrophy but not matrix expansion in the diabetic kidney. J. Cell Mol. Med. 15, 414-422). Because studies have also implicated the reactive oxygen species-generating enzymes NADPH oxidases (Nox) in diabetic kidney responses, we tested the hypothesis that Nox and Cn cooperate in a common signaling pathway. First, we examined the role of the two main isoforms of Cn in hypertrophic signaling. Using primary kidney cells lacking a catalytic subunit of Cn (CnA?(-/-) or CnA?(-/-)), we found that high glucose selectively activates CnA?, whereas CnA? is constitutively active. Furthermore, CnA? but not CnA? mediates hypertrophy. Next, we found that chronic reactive oxygen species generation in response to high glucose is attenuated in CnA?(-/-) cells, suggesting that Cn is upstream of Nox. Consistent with this, loss of CnA? reduces basal expression and blocks high glucose induction of Nox2 and Nox4. Inhibition of nuclear factor of activated T cells (NFAT), a CnA?-regulated transcription factor, decreases Nox2 and Nox4 expression, whereas NFAT overexpression increases Nox2 and Nox4, indicating that the CnA?/NFAT pathway modulates Nox. These data reveal that the CnA?/NFAT pathway regulates Nox and plays an important role in high glucose-mediated hypertrophic responses in the kidney. PMID:24371139

Williams, Clintoria R; Gooch, Jennifer L

2014-02-21

102

Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia.  

PubMed

We investigated the protective effect of electroacupuncture (EA) on cerebral ischemic injury in diabetic mice, and explored the role of NADPH oxidase-mediated oxidative stress. Male C57BL/6 mice were injected streptozotocin to induce diabetes. The mice were pretreated with EA at acupoint "Baihui" for 30 min. Two hours after the end of EA pretreatment, focal cerebral ischemia was induced following 24h reperfusion. The neurobehavioral scores and infarction volumes, malondialdehyde (MDA), reactive oxygen species (ROS), and activation of NADPH oxidase were determined in the presence or absence of the NADPH oxidase inhibitor apocynin or activator tetrabromocinnamic acid (TBCA). EA pretreatment reduced infarct size and improved neurological outcomes 24h after reperfusion in the diabetic mice. EA also decreased cerebral MDA and ROS levels compared with the control group, and inhibited the NADPH oxidase activation. The beneficial effects were abolished by TBCA while pretreatment with apocynin mimicked the neuroprotective and anti-oxidative effects of EA. Our results demonstrated that EA attenuated cerebral ischemic injury by inhibiting NAPDH oxidase-mediated oxidative damage in diabetic mice. These results suggest a novel mechanism of EA pretreatment-induced tolerance in diabetic cerebral ischemia. PMID:24854123

Guo, Fan; Song, Wenying; Jiang, Tao; Liu, Lixin; Wang, Feng; Zhong, Haixing; Yin, Hong; Wang, Qiang; Xiong, Lize

2014-07-21

103

Synaptic localization of a functional NADPH oxidase in the mouse hippocampus  

Microsoft Academic Search

Superoxide has been shown to be critical for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory function. A possible source for the generation of superoxide during these processes is NADPH oxidase. The active oxidase consists of two membrane proteins, gp91phox and p22phox, and four cytosolic proteins, p40phox, p47phox, p67phox, and Rac. Upon stimulation, the cytosolic proteins translocate to the membrane to

Maria V. Tejada-Simon; Faridis Serrano; Laura E. Villasana; Beatriz I. Kanterewicz; Gang-Yi Wu; Mark T. Quinn; Eric Klann

2005-01-01

104

NADPH Oxidases as Novel Pharmacologic Targets against Influenza A Virus Infection.  

PubMed

Influenza A viruses represent a major global health care challenge, with imminent pandemics, emerging antiviral resistance, and long lag times for vaccine development, raising a pressing need for novel pharmacologic strategies that ideally target the pathology irrespective of the infecting strain. Reactive oxygen species (ROS) pervade all facets of cell biology with both detrimental and protective properties. Indeed, there is compelling evidence that activation of the NADPH oxidase 2 (NOX2) isoform of the NADPH oxidase family of ROS-producing enzymes promotes lung oxidative stress, inflammation, injury, and dysfunction resulting from influenza A viruses of low to high pathogenicity, as well as impeding virus clearance. By contrast, the dual oxidase isoforms produce ROS that provide vital protective antiviral effects for the host. In this review, we propose that inhibitors of NOX2 are better alternatives than broad-spectrum antioxidant approaches for treatment of influenza pathologies, for which clinical efficacy may have been limited owing to poor bioavailability and inadvertent removal of beneficial ROS. Finally, we briefly describe the current suite of NADPH oxidase inhibitors and the molecular features of the NADPH oxidase enzymes that could be exploited by drug discovery for development of more specific and novel inhibitors to prevent or treat disease caused by influenza. PMID:25301784

Vlahos, Ross; Selemidis, Stavros

2014-12-01

105

NETosis and NADPH oxidase: at the intersection of host defense, inflammation, and injury  

PubMed Central

Neutrophils are armed with both oxidant-dependent and -independent pathways for killing pathogens. Activation of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase constitutes an emergency response to infectious threat and results in the generation of antimicrobial reactive oxidants. In addition, NADPH oxidase activation in neutrophils is linked to activation of granular proteases and generation of neutrophil extracellular traps (NETs). NETosis involves the release of nuclear and granular components that can target extracellular pathogens. NETosis is activated during microbial threat and in certain conditions mimicking sepsis, and can result in both augmented host defense and inflammatory injury. In contrast, apoptosis, the physiological form of neutrophil death, not only leads to non-inflammatory cell death but also contributes to alleviate inflammation. Although there are significant gaps in knowledge regarding the specific contribution of NETs to host defense, we speculate that the coordinated activation of NADPH oxidase and NETosis maximizes microbial killing. Work in engineered mice and limited patient experience point to varying susceptibility of bacterial and fungal pathogens to NADPH oxidase versus NET constituents. Since reactive oxidants and NET constituents can injure host tissue, it is important that these pathways be tightly regulated. Recent work supports a role for NETosis in both acute lung injury and in autoimmunity. Knowledge gained about mechanisms that modulate NETosis may lead to novel therapeutic approaches to limit inflammation-associated injury. PMID:23459634

Almyroudis, Nikolaos G.; Grimm, Melissa J.; Davidson, Bruce A.; Rohm, Marc; Urban, Constantin F.; Segal, Brahm H.

2013-01-01

106

Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones.  

PubMed

NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)-type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91(phox) localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40(phox) . p40(phox) itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91(phox) and p40(phox) with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40(phox) with NOX2/gp91(phox) at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. We have previously shown that reactive oxygen species (ROS) are critical for actin organization and dynamics in neuronal growth cones as well as neurite outgrowth. Here, we report that the cytosolic subunit p40(phox) of the NOX2-type NADPH oxidase complex is partially associated with F-actin in neuronal growth cones, while ROS produced by this complex regulates F-actin dynamics and neurite growth. These findings provide evidence for a bidirectional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones. PMID:24702317

Munnamalai, Vidhya; Weaver, Cory J; Weisheit, Corinne E; Venkatraman, Prahatha; Agim, Zeynep Sena; Quinn, Mark T; Suter, Daniel M

2014-08-01

107

Structural changes are induced in human neutrophil cytochrome b by NADPH oxidase activators, LDS, SDS, and arachidonate: intermolecular resonance energy transfer between trisulfopyrenyl-wheat germ agglutinin and cytochrome b 558  

Microsoft Academic Search

Anionic amphiphiles such as sodium- and lithium dodecyl sulfate (SDS, LDS), or arachidonate (AA) initiate NADPH oxidase and proton channel activation in cell-free systems and intact neutrophils. To investigate whether these amphiphiles exert allosteric effects on cytochrome b, trisulfopyrenyl-labeled wheat germ agglutinin (Cascade Blue®–wheat germ agglutinin, CCB-WGA) was used as an extrinsic fluorescence donor for resonance energy transfer (RET) to

Thomas R Foubert; James B Burritt; Ross M Taylor; Algirdas J Jesaitis

2002-01-01

108

Hace1 controls ROS generation of vertebrate Rac1-dependent NADPH oxidase complexes  

PubMed Central

The Hace1-HECT E3 ligase is a tumor suppressor that ubiquitylates the activated GTP-bound form of the Rho family GTPase Rac1, leading to Rac1 proteasomal degradation. Here we show that, in vertebrates, Hace1 targets Rac1 for degradation when Rac1 is localized to the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase holoenzyme. This event blocks de novo reactive oxygen species generation by Rac1-dependent NADPH oxidases, and thereby confers cellular protection from reactive oxygen species-induced DNA damage and cyclin D1-driven hyper-proliferation. Genetic inactivation of Hace1 in mice or zebrafish, as well as Hace1 loss in human tumor cell lines or primary murine or human tumors, leads to chronic NADPH oxidase-dependent reactive oxygen species elevation, DNA damage responses and enhanced cyclin D1 expression. Our data reveal a conserved ubiquitin-dependent molecular mechanism that controls the activity of Rac1-dependent NADPH oxidase complexes, and thus constitutes the first known example of a tumor suppressor protein that directly regulates reactive oxygen species production in vertebrates. PMID:23864022

Daugaard, Mads; Nitsch, Roberto; Razaghi, Babak; McDonald, Lindsay; Jarrar, Ameer; Torrino, Stéphanie; Castillo-Lluva, Sonia; Rotblat, Barak; Li, Liheng; Malliri, Angeliki; Lemichez, Emmanuel; Mettouchi, Amel; Berman, Jason N.; Penninger, Josef M.; Sorensen, Poul H.

2013-01-01

109

NADPH Oxidases Regulate CD44 and Hyaluronic Acid Expression in Thrombin-treated Vascular Smooth Muscle Cells and in Atherosclerosis*  

PubMed Central

The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE?/? mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ROS regulate the expression of CD44 and its principal ligand, hyaluronan (HA), and how CD44-HA interaction affects VSMC proliferation and migration and inflammatory gene expression in apoE?/? mice aortas. Thrombin-induced CD44 expression is mediated by transcription factor AP-1 in a NADPH oxidase-dependent manner. NADPH oxidase-mediated ROS generation enhanced thrombin-induced HA synthesis, and hyaluronan synthase 2 expression in VSMC. Hyaluronidase, which generates low molecular weight HA (LMW-HA), is induced in VSMC in a NADPH oxidase-dependent manner and LMW-HA stimulated ROS generation and cell proliferation in wild-type but not p47phox?/? VSMC, effects that were enhanced by thrombin pretreatment. Haptotactic VSMC migration toward HA was increased by thrombin in a CD44-dependent manner. HA expression in atherosclerotic lesions and plasma-soluble CD44 and HA levels were higher in apoE?/? compared with apoE?/?/p47phox?/? mice. HA-regulated pro-inflammatory gene expression was higher in apoE?/? than apoE?/?/p47phox?/? mouse aortas. GKT136901, a specific inhibitor of Nox1- and Nox4-containing NADPH oxidase activity, attenuated ROS generation and atherosclerosis and decreased CD44 and HA expression in atherosclerotic lesions. Together, these data suggest that increased CD44 and HA expression and CD44-HA-dependent gene regulation may play a role in atherosclerosis stimulated by NADPH oxidase activation. PMID:20558727

Vendrov, Aleksandr E.; Madamanchi, Nageswara R.; Niu, Xi-Lin; Molnar, Kimberly C.; Runge, Mason; Szyndralewiez, Cedric; Page, Patrick; Runge, Marschall S.

2010-01-01

110

NADPH oxidase 4 limits bone mass by promoting osteoclastogenesis  

PubMed Central

ROS are implicated in bone diseases. NADPH oxidase 4 (NOX4), a constitutively active enzymatic source of ROS, may contribute to the development of such disorders. Therefore, we studied the role of NOX4 in bone homeostasis. Nox4–/– mice displayed higher bone density and reduced numbers and markers of osteoclasts. Ex vivo, differentiation of monocytes into osteoclasts with RANKL and M-CSF induced Nox4 expression. Loss of NOX4 activity attenuated osteoclastogenesis, which was accompanied by impaired activation of RANKL-induced NFATc1 and c-JUN. In an in vivo model of murine ovariectomy–induced osteoporosis, pharmacological inhibition or acute genetic knockdown of Nox4 mitigated loss of trabecular bone. Human bone obtained from patients with increased osteoclast activity exhibited increased NOX4 expression. Moreover, a SNP of NOX4 was associated with elevated circulating markers of bone turnover and reduced bone density in women. Thus, NOX4 is involved in bone loss and represents a potential therapeutic target for the treatment of osteoporosis. PMID:24216508

Goettsch, Claudia; Babelova, Andrea; Trummer, Olivia; Erben, Reinhold G.; Rauner, Martina; Rammelt, Stefan; Weissmann, Norbert; Weinberger, Valeska; Benkhoff, Sebastian; Kampschulte, Marian; Obermayer-Pietsch, Barbara; Hofbauer, Lorenz C.; Brandes, Ralf P.; Schroder, Katrin

2013-01-01

111

NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy.  

PubMed

Objectives. Using apocynin (inhibitor of NADPH oxidase), and Mitoquinol 10 nitrate (MitoQ; mitochondrial-targeted antioxidant), we addressed the importance of mitochondria versus NADPH oxidase-derived ROS in glucose-induced apoptosis of pericytes. Methods. NADPH oxidase was localised using Western blot analysis and cytochrome C reduction assay. Apoptosis was detected by measuring caspase-3 activity. Intracellular glucose concentration, ROS formation and Nepsilon-(carboxymethyl) lysine (CML) content were measured using Amplex Red assay kit, dihydroethidium (DHE), and competitive immunoabsorbant enzyme-linked assay (ELISA), respectively. Results. NADPH oxidase was localised in the cytoplasm of pericytes suggesting ROS production within intracellular compartments. High glucose (25 mM) significantly increased apoptosis, intracellular glucose concentration, and CML content. Apoptosis was associated with increased gp91phox expression, activity of NADPH oxidase, and intracellular ROS production. Apocynin and not MitoQ significantly blunted the generation of ROS, formation of intracellular CML and apoptosis. Conclusions. NADPH oxidase and not mitochondria-derived ROS is responsible for the accelerated apoptosis of pericytes in diabetic retinopathy. PMID:20652059

Mustapha, Nik M; Tarr, Joanna M; Kohner, Eva M; Chibber, Rakesh

2010-01-01

112

Role of NADPH Oxidase-Mediated Reactive Oxygen Species in Podocyte Injury  

PubMed Central

Proteinuria is an independent risk factor for end-stage renal disease (ESRD) (Shankland, 2006). Recent studies highlighted the mechanisms of podocyte injury and implications for potential treatment strategies in proteinuric kidney diseases (Zhang et al., 2012). Reactive oxygen species (ROS) are cellular signals which are closely associated with the development and progression of glomerular sclerosis. NADPH oxidase is a district enzymatic source of cellular ROS production and prominently expressed in podocytes (Zhang et al., 2010). In the last decade, it has become evident that NADPH oxidase-derived ROS overproduction is a key trigger of podocyte injury, such as renin-angiotensin-aldosterone system activation (Whaley-Connell et al., 2006), epithelial-to-mesenchymal transition (Zhang et al., 2011), and inflammatory priming (Abais et al., 2013). This review focuses on the mechanism of NADPH oxidase-mediated ROS in podocyte injury under different pathophysiological conditions. In addition, we also reviewed the therapeutic perspectives of NADPH oxidase in kidney diseases related to podocyte injury. PMID:24319690

Chen, Shan; Meng, Xian-Fang; Zhang, Chun

2013-01-01

113

Respiratory burst and NAD(P)H oxidase activity are involved in capacitation of cryopreserved bovine spermatozoa  

Microsoft Academic Search

Heparin (a glycosaminoglycan) and quercetin (a calcium-ATPase plasma membrane specific inhibitor) induce bovine sperm capacitation. Mitochondria from frozen semen are capable of generating oxidative energy. The aim of the study was to determine oxygen uptake variation and the participation of diphenileneiodonium (DPI)-sensitive oxidases from spermatozoa capacitated with heparin or quercetin. Oxygen uptake was measured polarographically and 2?M diphenileneiodonium (DPI) was

M. Córdoba; N. Mora; M. T. Beconi

2006-01-01

114

PPAR? and NAD(P)H oxidase system interaction in glycerol-induced acute renal failure: role of gp91phox subunit of NAD(P)H oxidase.  

PubMed

Increased NAD(P)H oxidase-dependent free radical generation has been proposed to be a mechanism in glycerol-induced acute renal failure (ARF). Previously, we showed a PPAR?-mediated regulation of free radical generation in ARF. In this study, we examined NAD(P)H oxidase-dependent pathology in ARF and its connection with PPAR? using both Sprague-Dawley rats and gp91phox (+/-) mice. Male gp91phox (+/-) or wild type (+/+) mice were distributed into vehicle and ARF group (50% glycerol; 8 mL/kg bw; i.m.). Animals were placed in metabolic cages for 24 hr and were sacrificed under pentobarbital anesthesia. Urine, plasma and kidneys were processed for biochemical and molecular analysis. Glycerol doubled proteinuria in (+/+) mice (68 ± 4 mg/24 hr) but not in (+/-) mice (43 ± 9 mg/24 hr). This was associated with a markedly reduced creatinine excretion in (+/+) mice (Con: 0.6 ± 0.03 & ARF: 0.37 ± 0.02). Basal plasma and urinary NO was higher in (+/-) mice than the (+/+) type while plasma 8-isoprostane level was lower in (+/-) mice (WT: 165 ± 20; KO: 100 ± 15 pg/mL). Glycerol reduced UNOXV in both (+/+) and (+/-) mice although plasma NO was unchanged. Glycerol also doubled 8-isoprostane in (+/+) (363 ± 22 pg/mL) but not in (+/-) mice (152 ± 20 pg/mL) and this was associated with an increased NAD(P)H oxidase activity in the (+/+) mice. In ARF, PPAR? expression was reduced in (+/+) mice but increased in (+/-) mice. PPAR? activity was also reduced in (+/+) mice but was unchanged in (+/-) mice. We conclude that gp91phox contributes to NAD(P)H oxidase-mediated increased free radical generation in ARF and this may be via reduced PPAR?. PMID:24479849

Newaz, Mohammad; Yousefipour, Zivar

2014-05-01

115

Impact of Cyanidin-3-Glucoside on Glycated LDL-Induced NADPH Oxidase Activation, Mitochondrial Dysfunction and Cell Viability in Cultured Vascular Endothelial Cells  

PubMed Central

Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC. PMID:23443099

Xie, Xueping; Zhao, Ruozhi; Shen, Garry X.

2012-01-01

116

Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets  

Microsoft Academic Search

NADPH oxidases are a family of enzymes that generate reactive oxygen species (ROS). The NOX1 (NADPH oxidase 1) and NOX2 oxidases are the major sources of ROS in the artery wall in conditions such as hypertension, hypercholesterolaemia, diabetes and ageing, and so they are important contributors to the oxidative stress, endothelial dysfunction and vascular inflammation that underlies arterial remodelling and

Stavros Selemidis; Kathy K. Griendling; Christopher G. Sobey; Grant R. Drummond

2011-01-01

117

STIM1 but not STIM2 is an essential regulator of Ca2+ influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells.  

PubMed

Extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has been linked to various inflammatory diseases, regulatory mechanisms that control Ca2+ influx-induced NOX2 activation are poorly understood in SOCE. The role of STIM1, a Ca2+ sensor that transduces the store depletion signal to the plasma membrane, seems well established and supported by numerous studies in non-phagocytic cells. Here, in neutrophil-like HL-60 cells we used a siRNA approach to delineate the effect of STIM1 knock-down on NOX2 activity regulated by Ca2+ influx. Because the function of the STIM1 homolog, STIM2, is still unclear, we determined the consequence of STIM2 knock-down on Ca2+ and NOX2. STIM1 and STIM2 knock-down was effective and isoform specific when assayed by real-time PCR and Western blotting. Consistent with a unique role of STIM1 in the regulation of SOCE, STIM1, but not STIM2, siRNA significantly decreased Ca2+ influx induced by fMLF or the SERCA pump inhibitor thapsigargin. A redistribution of STIM1, originally localized intracellularly, near the plasma membrane was observed by confocal microscopy upon stimulation by fMLF. Inhibition of STIM1-induced SOCE led to a marked decrease in NOX2 activity while STIM2 siRNA had no effect. Thus, our results provide evidence for a role of STIM1 protein in the control of Ca2+ influx in neutrophils excluding a STIM2 involvement in this process. It also places STIM1 as a key modulator of NOX2 activity with a potential interest for anti-inflammatory pharmacological development. PMID:19433064

Bréchard, S; Plançon, S; Melchior, C; Tschirhart, E J

2009-09-01

118

Structural changes are induced in human neutrophil cytochrome b by NADPH oxidase activators, LDS, SDS, and arachidonate: intermolecular resonance energy transfer between trisulfopyrenyl-wheat germ agglutinin and cytochrome b(558).  

PubMed

Anionic amphiphiles such as sodium- and lithium dodecyl sulfate (SDS, LDS), or arachidonate (AA) initiate NADPH oxidase and proton channel activation in cell-free systems and intact neutrophils. To investigate whether these amphiphiles exert allosteric effects on cytochrome b, trisulfopyrenyl-labeled wheat germ agglutinin (Cascade Blue-wheat germ agglutinin, CCB-WGA) was used as an extrinsic fluorescence donor for resonance energy transfer (RET) to the intrinsic heme acceptors of detergent-solubilized cytochrome b. In solution, cytochrome b complexed with the CCB-WGA causing a rapid, saturable, carbohydrate-dependent quenching of up to approximately 55% of the steady-state fluorescence. Subsequent additions of SDS, LDS, or AA to typical cell-free oxidase assay concentrations completely relaxed the fluorescence quenching. The relaxation effects were specific, and not caused by dissociation of the CCB-WGA-cytochrome b complex or alterations in the spectral properties of the chromophores. In contrast, addition of the oxidase antagonist, arachidonate methyl ester, caused an opposite effect and was able to partially reverse the activator-induced relaxation. We conclude that the activators induce a cytochrome b conformation wherein the proximity or orientation between the hemes and the extrinsic CCB fluorescence donors has undergone a significant change. These events may be linked to NADPH oxidase assembly and activation or proton channel induction. PMID:12488056

Foubert, Thomas R; Burritt, James B; Taylor, Ross M; Jesaitis, Algirdas J

2002-12-23

119

Angiotensin II induces DNA damage via AT1 receptor and NADPH oxidase isoform Nox4.  

PubMed

Epidemiological studies revealed increased renal cancer incidences and higher cancer mortalities in hypertensive individuals. Activation of the renin-angiotensin-aldosterone system leads to the formation of reactive oxygen species (ROS). In vitro, in renal cells, and ex vivo, in the isolated perfused mouse kidney, we could show DNA-damaging potential of angiotensin II (Ang II). Here, the pathway involved in the genotoxicity of Ang II was investigated. In kidney cell lines with properties of proximal tubulus cells, an activation of NADPH oxidase and the production of ROS, resulting in the formation of DNA strand breaks and micronuclei induction, was observed. This DNA damage was mediated by the Ang II type 1 receptor (AT1R), together with the G protein G ( ?-q/11 ) . Subsequently, phospholipase C (PLC) was activated and intracellular calcium increased. Both calcium stores of the endoplasmic reticulum and extracellular calcium were involved in the genotoxicity of Ang II. Downstream, a role for protein kinase C (PKC) could be detected, because its inhibition hindered Ang II from damaging the cells. Although PKC was activated, no involvement of its known target, the NADPH oxidase isoform containing the Nox2 subunit, could be found, as tested by small-interfering RNA down-regulation. Responsible for the DNA-damaging activity of Ang II was the NADPH oxidase isoform containing the Nox4 subunit. In summary, in kidney cells the DNA-damaging activity of Ang II depends on an AT1R-mediated activation of NADPH oxidase via PLC, PKC and calcium signalling, with the NADPH subunit Nox4 playing a crucial role. PMID:22844079

Fazeli, Gholamreza; Stopper, Helga; Schinzel, Reinhard; Ni, Chih-Wen; Jo, Hanjoong; Schupp, Nicole

2012-11-01

120

Cytochrome Oxidase, Acetylcholinesterase, and NADPH-Diaphorase Staining in Human Supratemporal and Insular Cortex: Evidence for Multiple Auditory Areas  

Microsoft Academic Search

The pattern of cytochrome oxidase, acetylcholinesterase, and NADPH-diaphorase activity was studied in the supratemporal plane, the posterior part of the superior temporal gyrus, and the insula of normal human brains. Five dark cytochrome oxidase regions were found: (i) on Heschl's gyrus (area TC of von Economo and Koskinas); (ii) on the planum polare (area TC\\/TG); (iii) posterior to Heschl's gyrus

François Rivier; Stephanie Clarke

1997-01-01

121

Regulation of NADPH Oxidase 5 by Protein Kinase C Isoforms  

PubMed Central

NADPH oxidase5 (Nox5) is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKC? inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKC? and PKC?, enhanced by loss of PKC? and the silencing of PKC? expression was without effect. A constitutively active form of PKC? robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKC? potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKC? directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKC? activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKC? inhibitor, Go 6976. In summary, our study reveals that PKC? is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular complications of diabetes and other diseases with increased ROS production. PMID:24505490

Chen, Feng; Yu, Yanfang; Haigh, Steven; Johnson, John; Lucas, Rudolf; Stepp, David W.; Fulton, David J. R.

2014-01-01

122

Regulation of NADPH oxidase 5 by protein kinase C isoforms.  

PubMed

NADPH oxidase5 (Nox5) is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKC? inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKC? and PKC?, enhanced by loss of PKC? and the silencing of PKC? expression was without effect. A constitutively active form of PKC? robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKC? potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKC? directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKC? activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKC? inhibitor, Go 6976. In summary, our study reveals that PKC? is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular complications of diabetes and other diseases with increased ROS production. PMID:24505490

Chen, Feng; Yu, Yanfang; Haigh, Steven; Johnson, John; Lucas, Rudolf; Stepp, David W; Fulton, David J R

2014-01-01

123

Amyloid-? and Proinflammatory Cytokines Utilize a Prion Protein-Dependent Pathway to Activate NADPH Oxidase and Induce Cofilin-Actin Rods in Hippocampal Neurons  

PubMed Central

Neurites of neurons under acute or chronic stress form bundles of filaments (rods) containing 1?1 cofilin?actin, which impair transport and synaptic function. Rods contain disulfide cross-linked cofilin and are induced by treatments resulting in oxidative stress. Rods form rapidly (5–30 min) in >80% of cultured hippocampal or cortical neurons treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% of maximum response in ?6 h) occurs in a subpopulation (?20%) of hippocampal neurons upon exposure to soluble human amyloid-? dimer/trimer (A?d/t) at subnanomolar concentrations. Here we show that proinflammatory cytokines (TNF?, IL-1?, IL-6) also induce rods at the same rate and within the same neuronal population as A?d/t. Neurons from prion (PrPC)-null mice form rods in response to glutamate or antimycin A, but not in response to proinflammatory cytokines or A?d/t. Two pathways inducing rod formation were confirmed by demonstrating that NADPH-oxidase (NOX) activity is required for prion-dependent rod formation, but not for rods induced by glutamate or energy depletion. Surprisingly, overexpression of PrPC is by itself sufficient to induce rods in over 40% of hippocampal neurons through the NOX-dependent pathway. Persistence of PrPC-dependent rods requires the continuous activity of NOX. Removing inducers or inhibiting NOX activity in cells containing PrPC-dependent rods causes rod disappearance with a half-life of about 36 min. Cofilin-actin rods provide a mechanism for synapse loss bridging the amyloid and cytokine hypotheses for Alzheimer disease, and may explain how functionally diverse A?-binding membrane proteins induce synaptic dysfunction. PMID:24760020

Kane, Sarah J.; Shaw, Alisa E.; Brown, David R.; Pulford, Bruce; Zabel, Mark D.; Lambeth, J. David; Bamburg, James R.

2014-01-01

124

Amyloid-? and proinflammatory cytokines utilize a prion protein-dependent pathway to activate NADPH oxidase and induce cofilin-actin rods in hippocampal neurons.  

PubMed

Neurites of neurons under acute or chronic stress form bundles of filaments (rods) containing 1?1 cofilin?actin, which impair transport and synaptic function. Rods contain disulfide cross-linked cofilin and are induced by treatments resulting in oxidative stress. Rods form rapidly (5-30 min) in >80% of cultured hippocampal or cortical neurons treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% of maximum response in ?6 h) occurs in a subpopulation (?20%) of hippocampal neurons upon exposure to soluble human amyloid-? dimer/trimer (A?d/t) at subnanomolar concentrations. Here we show that proinflammatory cytokines (TNF?, IL-1?, IL-6) also induce rods at the same rate and within the same neuronal population as A?d/t. Neurons from prion (PrP(C))-null mice form rods in response to glutamate or antimycin A, but not in response to proinflammatory cytokines or A?d/t. Two pathways inducing rod formation were confirmed by demonstrating that NADPH-oxidase (NOX) activity is required for prion-dependent rod formation, but not for rods induced by glutamate or energy depletion. Surprisingly, overexpression of PrP(C) is by itself sufficient to induce rods in over 40% of hippocampal neurons through the NOX-dependent pathway. Persistence of PrP(C)-dependent rods requires the continuous activity of NOX. Removing inducers or inhibiting NOX activity in cells containing PrP(C)-dependent rods causes rod disappearance with a half-life of about 36 min. Cofilin-actin rods provide a mechanism for synapse loss bridging the amyloid and cytokine hypotheses for Alzheimer disease, and may explain how functionally diverse A?-binding membrane proteins induce synaptic dysfunction. PMID:24760020

Walsh, Keifer P; Minamide, Laurie S; Kane, Sarah J; Shaw, Alisa E; Brown, David R; Pulford, Bruce; Zabel, Mark D; Lambeth, J David; Kuhn, Thomas B; Bamburg, James R

2014-01-01

125

Does NAD(P)H oxidase-derived H2O2 participate in hypotonicity-induced insulin release by activating VRAC in ?-cells?  

PubMed

NAD(P)H oxidase (NOX)-derived H(2)O(2) was recently proposed to act, in several cells, as the signal mediating the activation of volume-regulated anion channels (VRAC) under a variety of physiological conditions. The present study aims at investigating whether a similar situation prevails in insulin-secreting BRIN-BD11 and rat ?-cells. Exogenous H(2)O(2) (100 to 200 ?M) at basal glucose concentration (1.1 to 2.8 mM) stimulated insulin secretion. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous H(2)O(2). In patch clamp experiments, exogenous H(2)O(2) was observed to stimulate NPPB-sensitive anion channel activity, which induced cell membrane depolarization. Exposure of the BRIN-BD11 cells to a hypotonic medium caused a detectable increase in intracellular level of reactive oxygen species (ROS) that was abolished by diphenyleneiodonium chloride (DPI), a universal NOX inhibitor. NOX inhibitors such as DPI and plumbagin nearly totally inhibited insulin release provoked by exposure of the BRIN-BD11 cells to a hypotonic medium. Preincubation with two other drugs also abolished hypotonicity-induced insulin release and reduced basal insulin output: 1) N-acetyl-L-cysteine (NAC), a glutathione precursor that serves as general antioxidant and 2) betulinic acid a compound that almost totally abolished NOX4 expression. As NPPB, each of these inhibitors (DPI, plumbagin, preincubation with NAC or betulinic acid) strongly reduced the volume regulatory decrease observed following a hypotonic shock, providing an independent proof that VRAC activation is mediated by H(2)O(2). Taken together, these data suggest that NOX-derived H(2)O(2) plays a key role in the insulin secretory response of BRIN-BD11 and native ?-cells to extracellular hypotonicity. PMID:22089811

Crutzen, R; Shlyonsky, V; Louchami, K; Virreira, M; Hupkens, E; Boom, A; Sener, A; Malaisse, W J; Beauwens, R

2012-02-01

126

The oxidation of apocynin catalyzed by myeloperoxidase: Proposal for NADPH oxidase inhibition  

Microsoft Academic Search

Apocynin has been used as an efficient inhibitor of the NADPH oxidase complex and its mechanism of inhibition is linked to prior activation through the action of peroxidases. Here we studied the oxidation of apocynin catalyzed by myeloperoxidase (MPO) and activated neutrophils. We found that apocynin is easily oxidized by MPO\\/H2O2 or activated neutrophils and has as products dimer and

Valdecir F. Ximenes; Marília P. P. Kanegae; Sandra R. Rissato; Mário S. Galhiane

2007-01-01

127

Oxidative innate immune defenses by Nox/Duox family NADPH oxidases.  

PubMed

The importance of reactive oxygen species (ROS) in innate immunity was first recognized in professional phagocytes undergoing a 'respiratory burst'upon activation. This robust oxygen consumption is related to a superoxide-generating enzyme, the phagocytic NADPH oxidase (Nox2-based or phox). The oxidase is essential for microbial killing, since patients lacking a functional oxidase suffer from enhanced susceptibility to microbial infections. ROS derived from superoxide attack bacteria in the isolated niche of the neutrophil phagosome. The oxidase is electrogenic, alters ion currents across membranes, induces apoptosis, regulates cytokine production, influences gene expression, and promotes formation of extracellular traps. Recently, new homologues of Nox2 were discovered establishing the Nox family of NADPH oxidases that encompasses seven members. Nox1 is highly expressed in the colon epithelium, and can be induced by LPS or IFN- gamma. Nox4 was implicated in innate immunity since LPS induces Nox4-dependent ROS generation. Duox1 and Duox2 localize to the apical plasma membrane of epithelial cells in major airways, salivary glands, and the gastrointestinal tract, and provide extracellular hydrogen peroxide to lactoperoxidase to produce antimicrobial hypothiocyanite ions. Th1 and Th2 cytokines regulate expression of dual oxidases in human airways and may thereby act in host defense or in proinflammatory responses. PMID:18511861

Rada, Balázs; Leto, Thomas L

2008-01-01

128

Angiotensin II, NADPH Oxidase, and Redox Signaling in the Vasculature  

PubMed Central

Abstract Significance: Angiotensin II (Ang II) influences the function of many cell types and regulates many organ systems, in large part through redox-sensitive processes. In the vascular system, Ang II is a potent vasoconstrictor and also promotes inflammation, hypertrophy, and fibrosis, which are important in vascular damage and remodeling in cardiovascular diseases. The diverse actions of Ang II are mediated via Ang II type 1 and Ang II type 2 receptors, which couple to various signaling molecules, including NADPH oxidase (Nox), which generates reactive oxygen species (ROS). ROS are now recognized as signaling molecules, critically placed in pathways activated by Ang II. Mechanisms linking Nox and Ang II are complex and not fully understood. Recent Advances: Ang II regulates vascular cell production of ROS through various recently characterized Noxs, including Nox1, Nox2, Nox4, and Nox5. Activation of these Noxs leads to ROS generation, which in turn influences many downstream signaling targets of Ang II, including MAP kinases, RhoA/Rho kinase, transcription factors, protein tyrosine phosphatases, and tyrosine kinases. Activation of these redox-sensitive pathways regulates vascular cell growth, inflammation, contraction, and senescence. Critical Issues: Although there is much evidence indicating a role for Nox/ROS in Ang II function, there is still a paucity of information on how Ang II exerts cell-specific effects through ROS and how Nox isoforms are differentially regulated by Ang II. Moreover, exact mechanisms whereby ROS induce oxidative modifications of signaling molecules mediating Ang II actions remain elusive. Future Directions: Future research should elucidate these issues to better understand the significance of Ang II and ROS in vascular (patho) biology. Antioxid. Redox Signal. 19, 1110–1120. PMID:22530599

Nguyen Dinh Cat, Aurelie; Montezano, Augusto C.; Burger, Dylan

2013-01-01

129

Subtractive screening reveals up-regulation of NADPH oxidase expression in Crohn's disease intestinal macrophages  

PubMed Central

Macrophages play a central role during the pathogenesis of inflammation. In normal intestinal mucosa surface expression of typical macrophage markers such as CD14, CD16, CD11b or T-cell co-stimulatory molecules such as CD80 or CD86 is low indicating anergy and low pro-inflammatory activity of these cells. During inflammatory bowel disease (IBD) the mucosa is invaded by a population of macrophages displaying these markers, secreting higher cytokine levels and representing an activated cell population. CD33+ cells (macrophages) were isolated from normal and Crohn's disease mucosa and mRNA was isolated by polyT magnetic beads. A subtractive screening was performed subtracting mRNA from normal macrophages from those of Crohn's disease macrophages. Oxidative burst activity was determined by flow cytometry. Seventy clones were obtained by the subtractive mRNA screening. Sequencing showed >99% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1) for three clones. Five clones obtained by subtraction revealed >99% homology to mRNA of cytochrome b (subunit gp91). Differential expression of the cytochrome b subunit gp91 and the cytosolic NADPH oxidase subunit p67 was confirmed by RT-PCR and ‘virtual’ Northern blots. The fluorescence ratio of stimulated versus unstimulated cells was 0·9 ± 0·16 in control macrophages indicating a lack of oxidative burst activity. In Crohn's disease this ratio was significantly increased to 1·80 ± 0·8 (P = 0·004) confirming the molecular data. In conclusion NADPH oxidase mRNA is down-regulated or absent in macrophages from normal mucosa correlating with a lack of oxidative burst activity. In IBD macrophage-oxidative burst activity is increased and NADPH oxidase mRNA induced. Inhibition of NADPH oxidase could be a new therapeutical target in IBD and reduce mucosal tissue damage in active IBD. PMID:11472425

Hausmann, M; Spottl, T; Andus, T; Rothe, G; Falk, W; Scholmerich, J; Herfarth, H; Rogler, G

2001-01-01

130

Fibroblast-to-myofibroblast switch is mediated by NAD(P)H oxidase generated reactive oxygen species  

PubMed Central

Tumour–stroma interaction is a prerequisite for tumour progression in skin cancer. Hereby, a critical step in stromal function is the transition of tumour-associated fibroblasts to MFs (myofibroblasts) by growth factors, for example TGF? (transforming growth factor beta(). In this study, the question was addressed of whether fibroblast-associated NAD(P)H oxidase (NADH/NADPH oxidase), known to be activated by TGF?1, is involved in the fibroblast-to-MF switch. The up-regulation of ?SMA (alpha smooth muscle actin), a biomarker for MFs, is mediated by a TGF?1-dependent increase in the intracellular level of ROS (reactive oxygen species). This report demonstrates two novel aspects of the TGF?1 signalling cascade, namely the generation of ROS due to a biphasic NAD(P)H oxidase activity and a ROS-dependent downstream activation of p38 leading to a transition of dermal fibroblasts to MFs that can be inhibited by the selective NAD(P)H oxidase inhibitor apocynin. These data suggest that inhibition of NAD(P)H oxidase activity prevents the fibroblast-to-MF switch and may be important for chemoprevention in context of a ‘stromal therapy’ which was described earlier. PMID:24299025

Alili, Lirija; Sack, Maren; Puschmann, Katharina; Brenneisen, Peter

2013-01-01

131

The roles of NADPH oxidase and phospholipases A2 in oxidative and inflammatory responses in neurodegenerative diseases.  

PubMed

Reactive oxygen species (ROS) are produced in mammalian cells through enzymic and non-enzymic mechanisms. Although some ROS production pathways are needed for specific physiological functions, excessive production is detrimental and is regarded as the basis of numerous neurodegenerative diseases. Among enzymes producing superoxide anions, NADPH oxidase is widespread in mammalian cells and is an important source of ROS in mediating physiological and pathological processes in the cardiovascular and the CNS. ROS production is linked to the alteration of intracellular calcium homeostasis, activation of Ca(2+)-dependent enzymes, alteration of cytoskeletal proteins, and degradation of membrane glycerophospholipids. There is evolving evidence that ROS produced by NADPH oxidase regulate neuronal functions and degrade membrane phospholipids through activation of phospholipases A(2) (PLA(2)). This review is intended to cover recent studies describing ROS generation from NADPH oxidase in the CNS and its downstream activation of PLA(2), namely, the group IV cytosolic cPLA(2) and the group II secretory sPLA(2). A major focus is to elaborate the dual role of NADPH oxidase and PLA(2) in mediating the oxidative and inflammatory responses in neurodegenerative diseases, including cerebral ischemia and Alzheimer's disease. Elucidation of the signaling pathways linking NADPH oxidase with the multiple forms of PLA(2) will be important in understanding the oxidative and degradative mechanisms that underline neuronal damage and glial activation and will facilitate development of therapeutic intervention for prevention and treatment of these and other neurodegenerative diseases. PMID:17561938

Sun, Grace Y; Horrocks, Lloyd A; Farooqui, Akhlaq A

2007-10-01

132

NADPH oxidase and the cardiovascular toxicity associated with smoking.  

PubMed

Smoking is one of the most serious but preventable causes of cardiovascular disease (CVD). Key aspects of pathological process associated with smoking include endothelial dysfunction, a prothrombotic state, inflammation, altered lipid metabolism, and hypoxia. Multiple molecular events are involved in smokinginduced CVD. However, the dysregulations of reactive oxygen species (ROS) generation and metabolism mainly contribute to the development of diverse CVDs, and NADPH oxidase (NOX) has been established as a source of ROS responsible for the pathogenesis of CVD. NOX activation and resultant ROS production by cigarette smoke (CS) treatment have been widely observed in isolated blood vessels and cultured vascular cells, including endothelial and smooth muscle cells. NOX-mediated oxidative stress has also been demonstrated in animal studies. Of the various NOX isoforms, NOX2 has been reported to mediate ROS generation by CS, but other isoforms were not tested thoroughly. Of the many CS constituents, nicotine, methyl vinyl ketone, and ?,?-unsaturated aldehydes, such as, acrolein and crotonaldehyde, appear to be primarily responsible for NOX-mediated cytotoxicity, but additional validation will be needed. Human epidemiological studies have reported relationships between polymorphisms in the CYBA gene encoding p22phox, a catalytic subunit of NOX and susceptibility to smoking-related CVDs. In particular, G allele carriers of A640G and -930(A/G) polymorphisms were found to be vulnerable to smoking-induced cardiovascular toxicity, but results for C242T studies are conflicting. On the whole, evidence implicates the etiological role of NOX in smoking-induced CVD, but the clinical relevance of NOX activation by smoking and its contribution to CVD require further validation in human studies. A detailed understanding of the role of NOX would be helpful to assess the risk of smoking to human health, to define high-risk subgroups, and to develop strategies to prevent or treat smoking-induced CVD. PMID:25343008

Kim, Mikyung; Han, Chang-Ho; Lee, Moo-Yeol

2014-09-01

133

NADPH Oxidase and the Cardiovascular Toxicity Associated with Smoking  

PubMed Central

Smoking is one of the most serious but preventable causes of cardiovascular disease (CVD). Key aspects of pathological process associated with smoking include endothelial dysfunction, a prothrombotic state, inflammation, altered lipid metabolism, and hypoxia. Multiple molecular events are involved in smokinginduced CVD. However, the dysregulations of reactive oxygen species (ROS) generation and metabolism mainly contribute to the development of diverse CVDs, and NADPH oxidase (NOX) has been established as a source of ROS responsible for the pathogenesis of CVD. NOX activation and resultant ROS production by cigarette smoke (CS) treatment have been widely observed in isolated blood vessels and cultured vascular cells, including endothelial and smooth muscle cells. NOX-mediated oxidative stress has also been demonstrated in animal studies. Of the various NOX isoforms, NOX2 has been reported to mediate ROS generation by CS, but other isoforms were not tested thoroughly. Of the many CS constituents, nicotine, methyl vinyl ketone, and ?,?-unsaturated aldehydes, such as, acrolein and crotonaldehyde, appear to be primarily responsible for NOX-mediated cytotoxicity, but additional validation will be needed. Human epidemiological studies have reported relationships between polymorphisms in the CYBA gene encoding p22phox, a catalytic subunit of NOX and susceptibility to smoking-related CVDs. In particular, G allele carriers of A640G and -930A/G polymorphisms were found to be vulnerable to smoking-induced cardiovascular toxicity, but results for C242T studies are conflicting. On the whole, evidence implicates the etiological role of NOX in smoking-induced CVD, but the clinical relevance of NOX activation by smoking and its contribution to CVD require further validation in human studies. A detailed understanding of the role of NOX would be helpful to assess the risk of smoking to human health, to define high-risk subgroups, and to develop strategies to prevent or treat smoking-induced CVD. PMID:25343008

Kim, Mikyung; Han, Chang-ho

2014-01-01

134

Rab27a regulates phagosomal pH and NADPH oxidase recruitment to dendritic cell phagosomes  

Microsoft Academic Search

To prevent excessive degradation of internalized antigens, which could destroy the peptides recognized by T lymphocytes, dendritic cells have developed several strategies that limit proteolytic activity in phagosomes. The recruitment of the NADPH oxidase NOX2 prevents acidification of phagosomes, limiting antigen degradation. Here, we show that dendritic cells derived from Rab27a-deficient ashen mice show increased phagosome acidification and antigen degradation,

Carolina Jancic; Ariel Savina; Christina Wasmeier; Tanya Tolmachova; Jamel El-Benna; Pham My-Chan Dang; Steve Pascolo; Maire-Anne Gougerot-Pocidalo; Graça Raposo; Miguel C. Seabra; Sebastian Amigorena

2007-01-01

135

Unconventional Roles of the NADPH Oxidase: Signaling, Ion Homeostasis, and Cell Death  

NSDL National Science Digital Library

Although the central role of the phagocytic NADPH oxidase in mediating bacterial killing has long been appreciated, this sophisticated enzyme complex serves various other important functions. This Perspective focuses on these underappreciated roles of phagocytic NADPH oxidase, highlighting recent work implicating reactive oxygen species in triggering an unconventional form of cell death.

Benjamin E. Steinberg (Hospital for Sick Children and University of Toronto;Program in Cell Biology REV); Sergio Grinstein (Hospital for Sick Children and University of Toronto;Program in Cell Biology REV)

2007-03-27

136

Tubulin ligands suggest a microtubule-NADPH oxidase relationship in postischemic cardiomyocytes.  

PubMed

Alterations of the microtubule network, which is involved in many vital processes, occur in several pathological conditions, such as cardiac ischemia. However, the connection between the microtubule assembly state and the factors affecting myocardial reperfusion injury, especially oxidative stress, is unknown. We aimed thus to study the effects of different tubulin ligands on the changes in the microtubule network and in several markers of cell injury and oxidative activity in cardiac muscle cells submitted to a reversible substrate-free, hypoxia-reoxygenation model of ischemia-reperfusion. The microtubule network was visualized by immunocytochemistry. Cell injury was evaluated via lactate dehydrogenase release and the mitochondrial function by the MTT test. Superoxide production was detected using dihydroethidium. The activity of NADPH oxidase and mRNA subunit expression were investigated. The microtubule disassembly induced by simulated ischemia was reversed by placing cardiomyocytes under normoxic conditions. This post-"ischemic" restoration of microtubule assembly was modulated by microtubule stabilizers (taxol: paclitaxel) and by microtubule disrupting drugs (nocodazole, colchicine). In addition, nocodazole decreased superoxide anion production as well as NADPH oxidase activity and mRNA expression of the NADPH oxidase subunit p22phox. These results demonstrated that the "ischemia"-induced microtubule network alteration is reversible and suggest a possible relationship between "reperfusion"-induced reassembly of microtubules and free radical generation in post-"ischemic" cardiomyocytes. PMID:16973157

Devillard, Lisa; Vandroux, David; Tissier, Cindy; Brochot, Amandine; Voisin, Sophie; Rochette, Luc; Athias, Pierre

2006-10-24

137

NADPH OXIDASES IN LUNG BIOLOGY AND PATHOLOGY. HOST DEFENSE ENZYMES, AND MORE  

PubMed Central

The deliberate production of reactive oxygen species (ROS) by phagocyte NADPH oxidase is widely appreciated as a critical component of antimicrobial host defense. Recently, additional homologs of NADPH oxidase (NOX) have been discovered throughout the animal and plant kingdoms, which appear to possess diverse functions in addition to host defense, including cell proliferation, differentiation, and regulation of gene expression. Several of these NOX homologs are also expressed within the respiratory tract, where they participate in innate host defense as well as in epithelial and inflammatory cell signaling and gene expression, and fibroblast and smooth muscle cell proliferation, in response to bacterial or viral infection and environmental stress. Inappropriate expression or activation of NOX/DUOX during various lung pathologies suggests their specific involvement in respiratory disease. This review summarizes the current state of knowledge regarding the general functional properties of mammalian NOX enzymes, and their specific importance in respiratory tract physiology and pathology. PMID:18164271

van der Vliet, Albert

2008-01-01

138

Soluble and insoluble immune complexes activate human neutrophil NADPH oxidase by distinct Fc gamma receptor-specific mechanisms.  

PubMed

Signal transduction initiated by interaction of immune complexes (IC) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied by investigating the capacity of well-defined complexes to stimulate O2- generation in neutrophils. IC consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complexes) and at five times Ag excess (soluble complexes). Stimulation of human neutrophils with soluble and insoluble IC caused a dose-dependent activation of the respiratory burst and O2- generation. Incubation of neutrophils with cytochalasin B significantly enhanced O2- generation in neutrophils stimulated with soluble IC. In contrast, cytochalasin B treatment had a minimal effect on O2- generation in neutrophils stimulated with insoluble IC. Treatment of neutrophils with PGE1 or pertussis toxin (PTx) significantly inhibited O2- generation by soluble IC-stimulated neutrophils. However, neither PGE1 nor PTx treatment significantly altered O2- generation in neutrophils stimulated with insoluble complexes. Although O2- generation induced by soluble IC was significantly inhibited by mAb against both Fc gamma RII and Fc gamma RIII receptor, insoluble IC stimulation of neutrophil O2- generation was significantly diminished only by mAb against Fc gamma RIII receptor. Cross-linking of either Fc gamma RII or Fc gamma RIII receptors on neutrophil surfaces induced O2- generation, and this activation was inhibited by both PGE1 and PTx treatment. These findings indicate that soluble and insoluble ICs induce O2- production in human neutrophils through distinct mechanisms. Soluble IC induce activation of neutrophils through a PTx- and PGE1-sensitive pathway that is dependent upon both Fc gamma RII and Fc gamma RIII receptors. Although insoluble IC induce O2- production through a PTx and PGE1 insensitive pathway mediated primarily through Fc gamma RIII receptor. PMID:2170530

Crockett-Torabi, E; Fantone, J C

1990-11-01

139

The Role of NADPH Oxidase in Chronic Intermittent Hypoxia-Induced Pulmonary Hypertension in Mice  

PubMed Central

Obstructive sleep apnea, characterized by intermittent periods of hypoxemia, is an independent risk factor for the development of pulmonary hypertension. However, the exact mechanisms of this disorder remain to be defined. Enhanced NADPH oxidase expression and superoxide (O2?·) generation in the pulmonary vasculature play a critical role in hypoxia-induced pulmonary hypertension. Therefore, the current study explores the hypothesis that chronic intermittent hypoxia (CIH) causes pulmonary hypertension, in part, by increasing NADPH oxidase–derived reactive oxygen species (ROS) that contribute to pulmonary vascular remodeling and hypertension. To test this hypothesis, male C57Bl/6 mice and gp91phox knockout mice were exposed to CIH for 8 hours per day, 5 days per week for 8 weeks. CIH mice were placed in a chamber where the oxygen concentration was cycled between 21% and 10% O2 45 times per hour. Exposure to CIH for 8 weeks increased right ventricular systolic pressure (RVSP), right ventricle (RV):left ventricle (LV) + septum (S) weight ratio, an index of RV hypertrophy, and thickness of the right ventricular anterior wall as measured by echocardiography. CIH exposure also caused pulmonary vascular remodeling as demonstrated by increased muscularization of the distal pulmonary vasculature. CIH-induced pulmonary hypertension was associated with increased lung levels of the NADPH oxidase subunits, Nox4 and p22phox, as well as increased activity of platelet-derived growth factor receptor ? and its associated downstream effector, Akt kinase. These CIH-induced derangements were attenuated in similarly treated gp91phox knockout mice. These findings demonstrate that NADPH oxidase–derived ROS contribute to the development of pulmonary vascular remodeling and hypertension caused by CIH. PMID:18952568

Nisbet, Rachel E.; Graves, Anitra S.; Kleinhenz, Dean J.; Rupnow, Heidi L.; Reed, Alana L.; Fan, Tai-Hwang M.; Mitchell, Patrick O.; Sutliff, Roy L.; Hart, C. Michael

2009-01-01

140

Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen.  

PubMed

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O(3)) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O(3) fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O(3) fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O(3), determined from the mRNA levels of the major allergens. We conclude that O(3) can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. PMID:21605929

Pasqualini, Stefania; Tedeschini, Emma; Frenguelli, Giuseppe; Wopfner, Nicole; Ferreira, Fatima; D'Amato, Gennaro; Ederli, Luisa

2011-10-01

141

Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen  

PubMed Central

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O3) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O3 fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O3 fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O3, determined from the mRNA levels of the major allergens. We conclude that O3 can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. PMID:21605929

Pasqualini, Stefania; Tedeschini, Emma; Frenguelli, Giuseppe; Wopfner, Nicole; Ferreira, Fatima; D'Amato, Gennaro; Ederli, Luisa

2011-01-01

142

Reactive Oxygen Species and Inhibitors of Inflammatory Enzymes, NADPH Oxidase, and iNOS in Experimental Models of Parkinson's Disease  

PubMed Central

Reactive oxygen species (ROSs) are emerging as important players in the etiology of neurodegenerative disorders including Parkinson's disease (PD). Out of several ROS-generating systems, the inflammatory enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and inducible nitric oxide synthase (iNOS) were believed to play major roles. Mounting evidence suggests that activation of NADPH oxidase and the expression of iNOS are directly linked to the generation of highly reactive ROS which affects various cellular components and preferentially damage midbrain dopaminergic neurons in PD. Therefore, appropriate management or inhibition of ROS generated by these enzymes may represent a therapeutic target to reduce neuronal degeneration seen in PD. Here, we have summarized recently developed agents and patents claimed as inhibitors of NADPH oxidase and iNOS enzymes in experimental models of PD. PMID:22577256

Koppula, Sushruta; Kumar, Hemant; Kim, In Su; Choi, Dong-Kug

2012-01-01

143

Redox-mediated signal transduction by cardiovascular Nox NADPH oxidases.  

PubMed

The only known function of the Nox family of NADPH oxidases is the production of reactive oxygen species (ROS). Some Nox enzymes show high tissue-specific expression and the ROS locally produced are required for synthesis of hormones or tissue components. In the cardiovascular system, Nox enzymes are low abundant and function as redox-modulators. By reacting with thiols, nitric oxide (NO) or trace metals, Nox-derived ROS elicit a plethora of cellular responses required for physiological growth factor signaling and the induction and adaptation to pathological processes. The interactions of Nox-derived ROS with signaling elements in the cardiovascular system are highly diverse and will be detailed in this article, which is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System". PMID:24560815

Brandes, Ralf P; Weissmann, Norbert; Schröder, Katrin

2014-08-01

144

[ROS and NADPH oxidase: key regulators of tumor vascularisation].  

PubMed

Oxidative stress is the result of an imbalance between the production of reactive oxygen species (ROS) and antioxidant mechanisms. It is characterized by damage of all cellular components, DNA, proteins, lipids. ROS are nevertheless important for the physiology of an organism, as they are involved in the innate immune defense and several intracellular signaling pathways. They play an important role in tumorigenesis by promoting tumor vasculature, which is essential to their growth and metastatic processes. There are many sources of ROS in the cells, but the NOX enzymes (NADPH oxidase-dependent) are now recognized to have a major role in the oxidative stress process. Indeed, they are present in many tissues where their only function is to produce ROS. This article discusses the NOX in endothelial cells and their role in the tumor angiogenesis. PMID:24801037

Garrido-Urbani, Sarah; Jaquet, Vincent; Imhof, Beat A

2014-04-01

145

Ginkgolide B attenuates ethanol-induced neurotoxicity through regulating NADPH oxidases.  

PubMed

Ethanol has long been demonstrated to trigger cell apoptosis in the central nervous system. The over-production of reactive oxygen species (ROS) is considered as one of the most important mechanisms involving in the apoptosis caused by ethanol. Ginkgolide B (GB), which was widely used as a monomer of traditional Chinese medicine, was reported to scavenge free radicals in endothelial cells and smooth muscle cells. But whether GB can prevent ethanol-induced neurotoxicity is still unknown. The aim of this study was to investigate effects of GB on ethanol-induced cytotoxicity, oxidative stress and apoptosis and explore potential protective molecular mechanism of GB. It was found that GB inhibited cell injury and apoptosis in a dose-dependent manner in ethanol-treated PC12 cells by MTT and LDH assays. It was also found that activities of caspase-3 increased by ethanol were mostly abrogated by GB. Further, GB decreased the production of ROS and subsequent over-production of lipid peroxides. A significant increase of alcohol dehydrogenase (ADH) and CYP2E1 enzyme activity was found in the ethanol-exposed PC12 cells as compared to controls. However, GB pretreatment did not significantly affect ethanol-induced ADH and CYP2E1 activities. Quantitative real-time PCR and Western blot analysis demonstrated that ethanol treatment resulted in a significant increase in mRNA and protein expression of NADPH oxidases, which are main oxidases producing ROS in neurons. Moreover, expression and activities of NADPH oxidases were down-regulated by GB. These results indicate that ethanol-induced neurotoxicity is ameliorated by GB mainly through regulating expression and activity of NADPH oxidases. PMID:21704112

Zhang, Chun; Tian, Xiujuan; Luo, Yi; Meng, Xianfang

2011-09-01

146

Assembly of NADPH oxidase in human neutrophils is modulated by the opacity-associated protein expression State of Neisseria gonorrhoeae.  

PubMed

Neisseria gonorrhoeae (the gonococcus, Gc) triggers a potent inflammatory response and recruitment of neutrophils to the site of infection. Gc survives exposure to neutrophils despite these cells' antimicrobial products, such as reactive oxygen species (ROS). ROS production in neutrophils is initiated by NADPH oxidase, which converts oxygen into superoxide. The subunits of NADPH oxidase are spatially separated between granules (gp91(phox)/p22(phox)) and the cytoplasm (p47(phox), p67(phox), and p40(phox)). Activation of neutrophils promotes the coassembly of NADPH oxidase subunits at phagosome and/or plasma membranes. While Gc-expressing opacity-associated (Opa) proteins can induce neutrophils to produce ROS, Opa-negative (Opa-) Gc does not stimulate neutrophil ROS production. Using constitutively Opa- and OpaD-positive (OpaD+) Gc bacteria in strain FA1090, we now show that the difference in ROS production levels in primary human neutrophils between these backgrounds can be attributed to differential assembly of NADPH oxidase. Neutrophils infected with Opa- Gc showed limited translocation of NADPH oxidase cytoplasmic subunits to cellular membranes, including the bacterial phagosome. In contrast, these subunits rapidly translocated to neutrophil membranes following infection with OpaD+ Gc. gp91(phox) and p22(phox) were recruited to Gc phagosomes regardless of bacterial Opa expression. These results suggest that Opa- Gc interferes with the recruitment of neutrophil NADPH oxidase cytoplasmic subunits to membranes, in particular, the p47(phox) "organizing" subunit, to prevent assembly of the holoenzyme, resulting in an absence of the oxidative burst. PMID:24343654

Smirnov, Asya; Daily, Kylene P; Criss, Alison K

2014-03-01

147

Regional Expression of NAD(P)H Oxidase and Superoxide Dismutase in the Brain of Rats with Neurogenic Hypertension  

Microsoft Academic Search

Background: Single injection of small quantities of phenol into the kidney cortex causes hypertension which is mediated by renal afferent sympathetic pathway activation. This phenomenon can be prevented by superoxide dismutase (SOD) infusion in the lateral ventricle, suggesting the role of superoxide (O2–·? ) in noradrenergic control of arterial pressure. Since NAD(P)H oxidase is a major source of O2–·? ,

Yongli Bai; Bahman Jabbari; Shaohua Ye; Vito M. Campese; Nosratola D. Vaziri

2009-01-01

148

Nox1Based NADPH Oxidase Is the Major Source of UVA-Induced Reactive Oxygen Species in Human Keratinocytes  

Microsoft Academic Search

UVA radiation is a major environmental stress on skin, causing acute and chronic photodamage. These responses are mediated by reactive oxygen species (ROS), although the cellular source of these ROS is unknown. We tested the hypotheses that UVA-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is required for ROS generation in human keratinocytes (HK) and that these ROS initiate

Antonio Valencia; Irene E Kochevar

2008-01-01

149

Nebivolol Reduces Proteinuria and Renal NADPH Oxidase-Generated Reactive Oxygen Species in the Transgenic Ren2 Rat  

PubMed Central

Background/Aims Renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system activation are crucial in the pathogenesis of hypertension, cardiovascular and renal disease. NADPH oxidase-mediated increases in reactive oxygen species (ROS) are an important mediator for RAAS-induced cardiovascular and renal injury. Increased levels of ROS can diminish the bioactivity of nitric oxide (NO), a critical modulator of RAAS effects on the kidney. Thereby, we hypothesized that in vivo nebivolol therapy in a rodent model of activated RAAS would attenuate glomerular damage and proteinuria through its actions to reduce NADPH oxidase activity/ROS and increase bioavailable NO. Methods We utilized the transgenic Ren2 rat which displays heightened tissue RAAS, hypertension, and proteinuria. Ren2 rats (6–9 weeks of age) and age-matched Sprague-Dawley littermates were treated with nebivolol 10 mg/kg/day (osmotic mini-pump) for 21 days. Results Ren2 rats exhibited increases in systolic blood pressure, proteinuria, kidney cortical tissue total NADPH oxidase activity and subunits (Rac1, p67phox, and p47phox), ROS and 3-nitrotyrosine, as well as reductions in podocyte protein markers; each of these parameters improved with nebivolol treatment along with increases in renal endothelial NO synthase expression. Conclusions Our data suggest that nebivolol improves proteinuria through reductions in renal RAAS-mediated increases in NADPH oxidase/ROS and increases in bioavailable NO. PMID:19609077

Whaley-Connell, Adam; Habibi, Javad; Johnson, Megan; Tilmon, Roger; Rehmer, Nathan; Rehmer, Jenna; Wiedmeyer, Charles; Ferrario, Carlos M.; Sowers, James R.

2009-01-01

150

The reactivity of ortho-methoxy-substituted catechol radicals with sulfhydryl groups: Contribution for the comprehension of the mechanism of inhibition of NADPH oxidase by apocynin  

Microsoft Academic Search

Redox processes are involved in the mechanism of action of NADPH oxidase inhibitors such as diphenyleneiodonium and apocynin. Here, we studied the structure-activity relationship for apocynin and analogous ortho-methoxy-substituted catechols as inhibitors of the NADPH oxidase in neutrophils and their reactivity with peroxidase. Aiming to alter the reduction potential, the ortho-methoxy-catechol moiety was kept constant and the substituents at para

Marília P. P. Kanegae; Luiz Marcos da Fonseca; Iguatemy L. Brunetti; Sueli de Oliveira Silva; Valdecir F. Ximenes

2007-01-01

151

Influence of NADPH oxidase on inflammatory response in primary intestinal epithelial cells in patients with ulcerative colitis  

PubMed Central

Background The aim of this study is to evaluate the role of NADPH oxidase in primary intestinal epithelial cells during the active phase of UC. Methods The primary human colonic epithelial cells were isolated from 19 patients with mild to moderate inflammatory activity of UC and 14 controls using chelation method. The cells were cultivated under the effect of mediators. Viability of cells was assessed by fluorescent microscopy. Production of reactive oxygen species (ROS) by the cells was measured fluorimetrically using Amplex Red. Production of TNF-? cytokine by the colonic epithelial cells was analysed by ELISA. Results The results of our study showed that unstimulated cells of UC patients had a decreased viability, increased ROS production, but similar TNF-? level when compared to the controls. Stimulation with LPS increased hydrogen peroxide and TNF-? level in the UC group. Treatment of colonic epithelial cells with NADPH oxidase inhibitor increased cell viability decreased the levels of ROS and TNF-? in the LPS-treated cells isolated from UC patients. Conclusions Our study showed that bacterial endotoxins induced NADPH oxidase activation in the colonic epithelial cells. Moreover, we revealed that treatment with NADPH oxidase inhibitors had a protective effect against pro-inflammatory action of LPS in human colonic epithelium cells during inflammation. PMID:24229374

2013-01-01

152

Oxidative stress in rats fed a high-fat high-sucrose diet and preventive effect of polyphenols: Involvement of mitochondrial and NAD(P)H oxidase systems.  

PubMed

Mitochondrial and NADPH oxidase systems and oxidative stress were investigated in 12 week high-fat high-sucrose (HFHS) diet-fed rats. A protective effect of wine polyphenol (PP) extract was also examined. In liver, maximal activities of CII and CII+III mitochondrial complexes were decreased but NADPH oxidase expression (p22(phox) and p47(phox)) and NADPH oxidase-dependent superoxide anion production were not modified, whereas oxidative stress (lipid and protein oxidation products and antioxidant systems) was increased with HFHS diet. In muscle, anion superoxide production was slightly increased while mitochondrial complex activities and lipid and protein oxidation products were not modified with HFHS diet. In heart, NADPH oxidase expression and superoxide anion production were increased, and maximal activity of mitochondrial respiratory chain complexes or oxidative stress parameters were not modified. Wine polyphenol extract had an inhibiting effect on liver oxidative stress and on heart NADPH oxidase expression and superoxide anion production, and on induction of hepatic steatosis with HFHS diet. Induction of mitochondrial dysfunction could be a primary event in the development of oxidative stress in liver, while in skeletal muscle and in heart the NADPH oxidase system seems to be mainly involved in oxidative stress. Wine polyphenol extract was shown to partially prevent oxidative stress in liver and heart tissues and to nearly completely prevent steatosis development in liver. PMID:19135522

Feillet-Coudray, C; Sutra, T; Fouret, G; Ramos, J; Wrutniak-Cabello, C; Cabello, G; Cristol, J P; Coudray, C

2009-03-01

153

NADPH Oxidase as a Therapeutic Target for Neuroprotection against Ischaemic Stroke: Future Perspectives  

PubMed Central

Oxidative stress caused by an excess of reactive oxygen species (ROS) is known to contribute to stroke injury, particularly during reperfusion, and antioxidants targeting this process have resulted in improved outcomes experimentally. Unfortunately these improvements have not been successfully translated to the clinical setting. Targeting the source of oxidative stress may provide a superior therapeutic approach. The NADPH oxidases are a family of enzymes dedicated solely to ROS production and pre-clinical animal studies targeting NADPH oxidases have shown promising results. However there are multiple factors that need to be considered for future drug development: There are several homologues of the catalytic subunit of NADPH oxidase. All have differing physiological roles and may contribute differentially to oxidative damage after stroke. Additionally, the role of ROS in brain repair is largely unexplored, which should be taken into consideration when developing drugs that inhibit specific NADPH oxidases after injury. This article focuses on the current knowledge regarding NADPH oxidase after stroke including in vivo genetic and inhibitor studies. The caution required when interpreting reports of positive outcomes after NADPH oxidase inhibition is also discussed, as effects on long term recovery are yet to be investigated and are likely to affect successful clinical translation. PMID:24961415

McCann, Sarah K.; Roulston, Carli L.

2013-01-01

154

NADPH oxidase mediates depressive behavior induced by chronic stress in mice.  

PubMed

Stress is a potent risk factor for depression, yet the underlying mechanism is not clearly understood. In the present study, we explored the mechanism of development and maintenance of depression in a stress-induced animal model. Mice restrained for 2 h daily for 14 d showed distinct depressive behavior, and the altered behavior persisted for >3 months in the absence of intervention. Acute restraint induced a surge of oxidative stress in the brain, and stress-induced oxidative stress progressively increased with repetition of stress. In vitro, the stress hormone glucocorticoid generated superoxide via upregulation of NADPH oxidase. Consistently, repeated restraints increased the expression of the key subunits of NADPH oxidase, p47phox and p67phox, in the brain. Moreover, stressed brains markedly upregulated the expression of p47phox to weak restress evoked in the poststress period, and this molecular response was reminiscent of amplified ROS surge to restress. Pharmacological inhibition of NADPH oxidase by the NADPH oxidase inhibitor apocynin during the stress or poststress period completely blocked depressive behavior. Consistently, heterozygous p47phox knock-out mice (p47phox(+/-)) or molecular inhibition of p47phox with Lenti shRNA-p47phox in the hippocampus suppressed depressive behavior. These results suggest that repeated stress promotes depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and that the inhibition of NADPH oxidase provides beneficial antidepression effects. PMID:22787054

Seo, Ji-Seon; Park, Jin-Young; Choi, Juli; Kim, Tae-Kyung; Shin, Joo-Hyun; Lee, Ja-Kyeong; Han, Pyung-Lim

2012-07-11

155

The NADPH-oxidase-associated H+ channel is opened by arachidonate.  

PubMed Central

The H+ channel associated with the generation of O2.- by NADPH oxidase and the oxidase itself must both be activated in response to stimuli (e.g. phorbol esters, chemotactic peptides, certain fatty acids). We have investigated the effects of membrane potential, an imposed pH gradient and a combination of the two (the protonmotive force) on the H+ conductivity of the cytoplast membrane. H+ conductivity was observed only in the presence of arachidonate and not in its absence. In the presence of arachidonate, H+ movement was determined by the protonmotive force. The effect of arachidonate was probably on a channel, since this fatty acid did not significantly increase the H+ permeability of artificial phospholipid membranes. It appears, therefore, that arachidonate is required both for the activation of O2.- production and the associated H(+)-channel-mediated efflux. PMID:1373602

Henderson, L M; Chappell, J B

1992-01-01

156

Nox2 NADPH oxidase promotes pathologic cardiac remodeling associated with Doxorubicin chemotherapy.  

PubMed

Doxorubicin is a highly effective cancer treatment whose use is severely limited by dose-dependent cardiotoxicity. It is well established that doxorubicin increases reactive oxygen species (ROS) production. In this study, we investigated contributions to doxorubicin cardiotoxicity from Nox2 NADPH oxidase, an important ROS source in cardiac cells, which is known to modulate several key processes underlying the myocardial response to injury. Nox2-deficient mice (Nox2-/-) and wild-type (WT) controls were injected with doxorubicin (12 mg/kg) or vehicle and studied 8 weeks later. Echocardiography indicated that doxorubicin-induced contractile dysfunction was attenuated in Nox2-/- versus WT mice (fractional shortening: 29.5±1.4 versus 25.7±1.0%; P<0.05). Similarly, in vivo pressure-volume analysis revealed that systolic and diastolic function was preserved in doxorubicin-treated Nox2-/- versus WT mice (ejection fraction: 52.6±2.5 versus 28.5±2.3%, LVdP/dtmin: -8,379±416 versus -5,198±527 mmHg s(-1); end-diastolic pressure-volume relation: 0.051±0.009 versus 0.114±0.012; P<0.001). Furthermore, in response to doxorubicin, Nox2-/- mice exhibited less myocardial atrophy, cardiomyocyte apoptosis, and interstitial fibrosis, together with reduced increases in profibrotic gene expression (procollagen III?I, transforming growth factor-?3, and connective tissue growth factor) and matrix metalloproteinase-9 activity, versus WT controls. These alterations were associated with beneficial changes in NADPH oxidase activity, oxidative/nitrosative stress, and inflammatory cell infiltration. We found that adverse effects of doxorubicin were attenuated by acute or chronic treatment with the AT1 receptor antagonist losartan, which is commonly used to reduce blood pressure. Our findings suggest that ROS specifically derived from Nox2 NADPH oxidase make a substantial contribution to several key processes underlying development of cardiac contractile dysfunction and remodeling associated with doxorubicin chemotherapy. PMID:20884632

Zhao, Youyou; McLaughlin, Declan; Robinson, Emma; Harvey, Adam P; Hookham, Michelle B; Shah, Ajay M; McDermott, Barbara J; Grieve, David J

2010-11-15

157

Apocynin, an NADPH oxidase inhibitor, suppresses rat prostate carcinogenesis.  

PubMed

Recent evidence suggests that oxidative stress contributes to the pathogenesis of prostate cancer. The present study focused on the effect of apocynin, an inhibitor of NADPH oxidase, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model. There were no toxic effects with apocynin treatment. The percentages and numbers of carcinomas in both the ventral and lateral prostate were significantly reduced by apocynin treatment, with dose dependence. Reduction of reactive oxygen species by apocynin was confirmed by immunohistochemistry of 8-OHdG and dihydroethidium staining. Positivity of Ki67 was significantly reduced by apocynin treatment, and downregulation of clusterin expression, as well as inactivation of the MEK-ERK1/2 pathway, was a feature of the apocynin treated groups. In human prostate cancer cell line LNCaP, apocynin also inhibited reactive oxygen species production and blocked cell growth by inducing G0/G1 arrest with downregulation of clusterin and cyclin D1. These data suggest that apocynin possesses chemopreventive potential against prostate cancer. PMID:24118288

Suzuki, Shugo; Shiraga, Kazuhide; Sato, Shinya; Punfa, Wanisa; Naiki-Ito, Aya; Yamashita, Yoriko; Shirai, Tomoyuki; Takahashi, Satoru

2013-12-01

158

Curcumin ameliorated diabetic neuropathy partially by inhibition of NADPH oxidase mediating oxidative stress in the spinal cord.  

PubMed

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are the main enzymes that produce oxidative stress, which plays an important role in painful diabetic neuropathy. Curcumin has been reported to exert an antinociceptive effect in a rat model of diabetic neuropathy by suppressing oxidative stress in the spinal cord. However, it remains unknown whether the mechanism by which curcumin ameliorates diabetic neuropathy can be attributed to spinal NADPH oxidases. This study was designed to determine the effect of curcumin on diabetic neuropathy and to investigate its precise mechanism in relation to NADPH oxidase-mediating oxidative stress in the spinal cord. Diabetic neuropathy was induced in Sprague-Dawley rats by intraperitoneal injection with 1% streptozotocin (STZ; 60 mg/kg). After the onset of diabetic neuropathy, a subset of the diabetic rats received daily intragastric administrations of curcumin (200mg/kg) or intraperitoneal injections of apocynin (2.5mg/kg) for 14 consecutive days, whereas other diabetic rats received equivalent volumes of normal saline (NS). STZ resulted in diabetic neuropathy with hyperglycemia and a lower paw withdrawal threshold (PWT), accompanied by elevations in the expression of the NADPH oxidase subunits p47(phox) and gp91(phox) and in the levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA) and a reduction in superoxide dismutase (SOD) activity (P<0.05) in the spinal cord. Both curcumin and apocynin ameliorated diabetic neuropathy. In conclusion, curcumin attenuated neuropathic pain in diabetic rats, at least partly by inhibiting NADPH oxidase-mediating oxidative stress in the spinal cord. PMID:24370596

Zhao, Wei-Cheng; Zhang, Bin; Liao, Mei-Juan; Zhang, Wen-Xuan; He, Wan-You; Wang, Han-Bing; Yang, Cheng-Xiang

2014-02-01

159

NADPH oxidase 1, mediates alpha-synucleinopathy in Parkinson's disease  

PubMed Central

Accumulation of misfolded ?-synuclein is the pathological hallmark of Parkinson’s disease (PD). Nevertheless, little is known about the mechanism contributing to ?-synuclein aggregation and its further toxicity to dopaminergic neurons. Since oxidative stress can increase the expression and aggregation levels of ?-synuclein, NADPH oxidases (NOXs), which are responsible for ROS generation, could be major players in ?-synucleinopathy. Previously, we demonstrated that Nox1 is expressed in dopaminergic neurons of the PD animal models as well as postmortem brain tissue of PD patients, being responsible for oxidative stress and subsequent neuronal degeneration. Here, using paraquat (PQ)-based in vitro and in vivo PD models, we show that Nox1 has a crucial role in modulating the behavior of ?-synuclein expression and aggregation in dopaminergic neurons. We observed in differentiated human dopaminergic cells that Nox1 and ?-synuclein expression are increased under PQ exposure. Nox1 knockdown significantly reduced both ?-synuclein expression and aggregation, supporting the role of Nox1 in this process. Furthermore, in rats exposed to PQ, the selective knockdown of Nox1 in the substantia nigra, using adeno-associated virus encoding Nox1-specific shRNA, largely attenuated the PQ-mediated increase of ?-synuclein and ubiquitin expression levels as well as ?-synuclein aggregates (proteinase-K resistant) and A11 oligomers. Significant reductions in oxidative stress level and dopaminergic neuronal loss were also observed. Our data reveal a new mechanism by which ?-synuclein becomes a neuropathologic protein through Nox1-mediated oxidative stress. This finding may be used to generate new therapeutic interventions that slower the rate of ?-synuclein aggregation and the progression of PD pathogenesis. PMID:23077033

Cristovao, Ana Clara; Guhathakurta, Subhrangshu; Bok, Eugene; Je, Goun; Yoo, Seung Don; Choi, Dong-Hee; Kim, Yoon-Seong

2012-01-01

160

NADPH Oxidase as a Therapeutic Target for Oxalate Induced Injury in Kidneys  

PubMed Central

A major role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes is to catalyze the production of superoxides and other reactive oxygen species (ROS). These ROS, in turn, play a key role as messengers in cell signal transduction and cell cycling, but when they are produced in excess they can lead to oxidative stress (OS). Oxidative stress in the kidneys is now considered a major cause of renal injury and inflammation, giving rise to a variety of pathological disorders. In this review, we discuss the putative role of oxalate in producing oxidative stress via the production of reactive oxygen species by isoforms of NADPH oxidases expressed in different cellular locations of the kidneys. Most renal cells produce ROS, and recent data indicate a direct correlation between upregulated gene expressions of NADPH oxidase, ROS, and inflammation. Renal tissue expression of multiple NADPH oxidase isoforms most likely will impact the future use of different antioxidants and NADPH oxidase inhibitors to minimize OS and renal tissue injury in hyperoxaluria-induced kidney stone disease. PMID:23840917

Peck, Ammon B.; Khan, Saeed R.

2013-01-01

161

NADPH oxidase and nitric oxide synthase dependent superoxide production is increased in proliferative lupus nephritis  

PubMed Central

Objective Lupus nephritis (LN) is an immune complex-mediated glomerulonephritis. Proliferative LN (PLN, International Society of Nephrology and Renal Pathology Society (ISN/RPS) classes III and IV)) often leads to renal injury or failure despite traditional induction and maintenance therapy. Successful targeted therapeutic development requires insight into mediators of inflammation in PLN. Superoxide (SO) and its metabolites are mediators of the innate immune response through their ability to mediate reduction-oxidation signaling. Endothelial nitric oxide synthase (eNOS) modulates inflammatory responses in endothelial cells. We hypothesized that markers of SO production would be increased in active PLN and that SO production would be dependent on the activity of select enzymes in the renal cortex. Methods Patients with systemic lupus erythematosus were enrolled at the time of renal biopsy for active LN of all classes. Serum collected at baseline was analyzed by HPLC with electrochemical detection for markers of SO production (durable modifications of serum protein Tyr ultimately requiring SO as a substrate). Renal cortex from MRL/MpJ-FASlpr (MRL/lpr) mice with and without functional eNOS was analyzed during active disease for superoxide (SO) production with and without inhibitors of SO producing enzymes. Results Serum protein modifications indicative of total SO production were significantly higher in patients with PLN. These markers were increased in association with more active, inflammatory PLN. Mice lacking functional eNOS had 80% higher levels of renal cortical SO during active disease, and inhibitors of nitric oxide synthase and NADPH oxidase reduced these levels by 60% and 77%, respectively. Conclusion These studies demonstrate that SO production is unique to active PLN in a NOS and NADPH oxidase-dependent fashion. These findings suggest the emulating or augmenting eNOS activity or inhibiting NADPH oxidase SO production may be targets of therapy in patients with PLN. The markers of SO production used in this study could rationally be used to select SO-modulating therapies and serve as pharmacodynamic indicators for dose titration. PMID:24106214

Oates, Jim C.; Mashmoushi, Ahmad K.; Shaftman, Stephanie R.; Gilkeson, Gary S.

2013-01-01

162

Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells  

PubMed Central

Background Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Methods Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. Results In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. Conclusions In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an increase in [Ca2+]i, P38 activation, and ROS generation via NADPH oxidase and mitochondria. PMID:25012725

2014-01-01

163

Interactions between NADPH oxidase-related proton and electron currents in human eosinophils  

PubMed Central

Proton and electron currents in human eosinophils were studied using the permeabilized-patch voltage-clamp technique, with an applied NH4+ gradient to control pHi.Voltage-gated proton channels in unstimulated human eosinophils studied with the permeabilized-patch approach had properties similar to those reported in whole-cell studies.Superoxide anion (O2?) release assessed by cytochrome c reduction was compared in human eosinophils and neutrophils stimulated by phorbol myristate acetate (PMA). PMA-stimulated O2 release was more transient and the maximum rate was three times greater in eosinophils.In PMA-activated eosinophils, the H+ current amplitude (IH) at +60 mV increased 4.7-fold, activation was 4.0 times faster, deactivation (tail current decay) was 5.4 times slower, the H+ conductance–voltage (gH–V) relationship was shifted ?43 mV, and diphenylene iodinium (DPI)-inhibitable inward current reflecting electron flow through NADPH oxidase was activated. The data reveal that PMA activates the H+ efflux during the respiratory burst by modulating the properties of H+ channels, not simply as a result of NADPH oxidase activity.The electrophysiological response of eosinophils to PMA resembled that reported in human neutrophils, but PMA activated larger proton and electron currents in eosinophils and the response was more transient.ZnCl2 slowed the activation of H+ currents and shifted the gH–V relationship to more positive voltages. These effects occurred at similar ZnCl2 concentrations in eosinophils before and after PMA stimulation. These data are compatible with the existence of a single type of H+ channel in eosinophils that is modulated during the respiratory burst. PMID:11559774

DeCoursey, T E; Cherny, V V; DeCoursey, A G; Xu, W; Thomas, L L

2001-01-01

164

Normalizing NADPH Oxidase Contributes to Attenuating Diabetic Nephropathy by the Dual Endothelin Receptor Antagonist CPU0213 in Rats  

Microsoft Academic Search

Background\\/Aims: NADPH oxidase(NOX) is the main source of reactive oxygen species (ROS) in diabetic nephropathy (DN). Activation of NOX could be mediated via endothelin A (ETAR) and B receptors (ETBR) of the endothelin (ET) system. Thus, CPU0213, a dual ET receptor antagonist, was expected to attenuate DN by suppressing NOX. Methods: Diabetes was produced in male Sprague-Dawley rats 8 weeks

Ming Xu; De-Zai Dai; Yin Dai

2009-01-01

165

Tumor necrosis factor-?-induced colitis increases NADPH oxidase 1 expression, oxidative stress, and neutrophil recruitment in the colon: preventive effect of apocynin.  

PubMed

Reactive oxygen species- (ROS-) mediated injury has been implicated in several inflammatory disorders, including inflammatory bowel disease (IBD). NADPH oxidases (NOXs) are the major source of endogenous ROS. Here, we investigated the role of NOXs derived-ROS in a mouse model of colitis induced by the proinflammatory cytokine, tumor necrosis factor-? (TNF-?). Intraperitoneal injection of TNF? (10 ?g · kg(-1)) induced an acute inflammation of the colon and a marked increase in expression of NADPH oxidase 1 (NOX1), a colon specific NADPH oxidase isoform. TNF?-induced colitis was also characterized by high production of keratinocyte-derived chemokine (KC) and mucosal infiltration of neutrophils, NOX2-expressing cells. Concomitantly, ROS production and lipid peroxidation were significantly enhanced while catalase activity and glutathione level were reduced indicating a redox imbalance in the colon. Furthermore, the redox-sensitive MAP kinases, ERK1/2 and p38 MAPK, were activated during TNF?-induced colitis. Pretreatment of mice with apocynin, an NADPH oxidase inhibitor with antioxidant properties, before TNF? challenge, prevented all these events. These data suggest that ROS derived from NADPH oxidases (mainly NOX1 and NOX2) and MAP kinase pathways could contribute to the induction and expansion of oxidative lesions characteristics of IBD and that apocynin could potentially be beneficial in IBD treatment. PMID:25276054

Mouzaoui, Souad; Djerdjouri, Bahia; Makhezer, Nesrine; Kroviarski, Yolande; El-Benna, Jamel; Dang, Pham My-Chan

2014-01-01

166

Tumor Necrosis Factor-?-Induced Colitis Increases NADPH Oxidase 1 Expression, Oxidative Stress, and Neutrophil Recruitment in the Colon: Preventive Effect of Apocynin  

PubMed Central

Reactive oxygen species- (ROS-) mediated injury has been implicated in several inflammatory disorders, including inflammatory bowel disease (IBD). NADPH oxidases (NOXs) are the major source of endogenous ROS. Here, we investigated the role of NOXs derived-ROS in a mouse model of colitis induced by the proinflammatory cytokine, tumor necrosis factor-? (TNF-?). Intraperitoneal injection of TNF? (10??g · kg?1) induced an acute inflammation of the colon and a marked increase in expression of NADPH oxidase 1 (NOX1), a colon specific NADPH oxidase isoform. TNF?-induced colitis was also characterized by high production of keratinocyte-derived chemokine (KC) and mucosal infiltration of neutrophils, NOX2-expressing cells. Concomitantly, ROS production and lipid peroxidation were significantly enhanced while catalase activity and glutathione level were reduced indicating a redox imbalance in the colon. Furthermore, the redox-sensitive MAP kinases, ERK1/2 and p38 MAPK, were activated during TNF?-induced colitis. Pretreatment of mice with apocynin, an NADPH oxidase inhibitor with antioxidant properties, before TNF? challenge, prevented all these events. These data suggest that ROS derived from NADPH oxidases (mainly NOX1 and NOX2) and MAP kinase pathways could contribute to the induction and expansion of oxidative lesions characteristics of IBD and that apocynin could potentially be beneficial in IBD treatment. PMID:25276054

Mouzaoui, Souad; Djerdjouri, Bahia; Makhezer, Nesrine; Kroviarski, Yolande; El-Benna, Jamel; Dang, Pham My-Chan

2014-01-01

167

FFA-induced hepatic insulin resistance in vivo is mediated by PKC?, NADPH oxidase, and oxidative stress.  

PubMed

Fat-induced hepatic insulin resistance plays a key role in the pathogenesis of type 2 diabetes in obese individuals. Although PKC and inflammatory pathways have been implicated in fat-induced hepatic insulin resistance, the sequence of events leading to impaired insulin signaling is unknown. We used Wistar rats to investigate whether PKC? and oxidative stress play causal roles in this process and whether this occurs via IKK?- and JNK-dependent pathways. Rats received a 7-h infusion of Intralipid plus heparin (IH) to elevate circulating free fatty acids (FFA). During the last 2 h of the infusion, a hyperinsulinemic-euglycemic clamp with tracer was performed to assess hepatic and peripheral insulin sensitivity. An antioxidant, N-acetyl-L-cysteine (NAC), prevented IH-induced hepatic insulin resistance in parallel with prevention of decreased I?B? content, increased JNK phosphorylation (markers of IKK? and JNK activation, respectively), increased serine phosphorylation of IRS-1 and IRS-2, and impaired insulin signaling in the liver without affecting IH-induced hepatic PKC? activation. Furthermore, an antisense oligonucleotide against PKC? prevented IH-induced phosphorylation of p47(phox) (marker of NADPH oxidase activation) and hepatic insulin resistance. Apocynin, an NADPH oxidase inhibitor, prevented IH-induced hepatic and peripheral insulin resistance similarly to NAC. These results demonstrate that PKC?, NADPH oxidase, and oxidative stress play a causal role in FFA-induced hepatic insulin resistance in vivo and suggest that the pathway of FFA-induced hepatic insulin resistance is FFA ? PKC? ? NADPH oxidase and oxidative stress ? IKK?/JNK ? impaired hepatic insulin signaling. PMID:24824652

Pereira, Sandra; Park, Edward; Mori, Yusaku; Haber, C Andrew; Han, Ping; Uchida, Toyoyoshi; Stavar, Laura; Oprescu, Andrei I; Koulajian, Khajag; Ivovic, Alexander; Yu, Zhiwen; Li, Deling; Bowman, Thomas A; Dewald, Jay; El-Benna, Jamel; Brindley, David N; Gutierrez-Juarez, Roger; Lam, Tony K T; Najjar, Sonia M; McKay, Robert A; Bhanot, Sanjay; Fantus, I George; Giacca, Adria

2014-07-01

168

Potential role of NADPH oxidase in pathogenesis of pancreatitis  

PubMed Central

Studies have demonstrated that reactive oxygen species (ROS) are closely related to inflammatory disorders. Nicotinamide adenine dinucleotide phosphate oxidase (NOX), originally found in phagocytes, is the main source of ROS in nonphagocytic cells. Besides directly producing the detrimental highly reactive ROS to act on biomolecules (lipids, proteins, and nucleic acids), NOX can also activate multiple signal transduction pathways, which regulate cell growth, proliferation, differentiation and apoptosis by producing ROS. Recently, research on pancreatic NOX is no longer limited to inflammatory cells, but extends to the aspect of pancreatic acinar cells and pancreatic stellate cells, which are considered to be potentially associated with pancreatitis. In this review, we summarize the literature on NOX protein structure, activation, function and its role in the pathogenesis of pancreatitis. PMID:25133019

Cao, Wei-Li; Xiang, Xiao-Hui; Chen, Kai; Xu, Wei; Xia, Shi-Hai

2014-01-01

169

NADPH oxidase 4 deficiency reduces aquaporin-2 mRNA expression in cultured renal collecting duct principal cells via increased PDE3 and PDE4 activity.  

PubMed

The final control of renal water reabsorption occurs in the collecting duct (CD) and relies on regulated expression of aquaporin-2 (AQP2) in principal CD cells. AQP2 transcription is primarily induced by type 2 vasopressin receptor (V2R)-cAMP-protein kinase A (PKA) signaling but also by other factors, including TonEBP and NF-?B. NAPDH oxidase 4 (NOX4) represents a major source of reactive oxygen species (ROS) in the kidney. Because NOX-derived ROS may alter PKA, TonEBP and NF-?B activity, we examined the effects of NOX4 depletion on AQP2 expression. Depleted NOX4 expression by siRNA (siNOX4) in mpkCCDcl4 cells attenuated increased AQP2 mRNA expression by arginine vasopressin (AVP) but not by hypertonicity, which induces both TonEBP and NF-?B activity. AVP-induced AQP2 expression was similarly decreased by the flavoprotein inhibitor diphenyleneiodonium. siNOX4 altered neither TonEBP nor NF-?B activity but attenuated AVP-inducible cellular cAMP concentration, PKA activity and CREB phosphorylation as well as AQP2 mRNA expression induced by forskolin, a potent activator of adenylate cyclase. The repressive effect of siNOX4 on AVP-induced AQP2 mRNA expression was abolished by the non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and was significantly decreased by selective PDE antagonists cilostamide and rolipram, but not vinpocetine, which respectively target PDE3, PDE4 and PDE1. Thus, by inhibiting PDE3 and PDE4 activity NOX4-derived ROS may contribute to V2R-cAMP-PKA signaling and enhance AQP2 transcription. PMID:24466344

Féraille, Eric; Dizin, Eva; Roth, Isabelle; Derouette, Jean-Paul; Szanto, Ildiko; Martin, Pierre-Yves; de Seigneux, Sophie; Hasler, Udo

2014-01-01

170

NADPH Oxidase 4 Deficiency Reduces Aquaporin-2 mRNA Expression in Cultured Renal Collecting Duct Principal Cells via Increased PDE3 and PDE4 Activity  

PubMed Central

The final control of renal water reabsorption occurs in the collecting duct (CD) and relies on regulated expression of aquaporin-2 (AQP2) in principal CD cells. AQP2 transcription is primarily induced by type 2 vasopressin receptor (V2R)-cAMP-protein kinase A (PKA) signaling but also by other factors, including TonEBP and NF-?B. NAPDH oxidase 4 (NOX4) represents a major source of reactive oxygen species (ROS) in the kidney. Because NOX-derived ROS may alter PKA, TonEBP and NF-?B activity, we examined the effects of NOX4 depletion on AQP2 expression. Depleted NOX4 expression by siRNA (siNOX4) in mpkCCDcl4 cells attenuated increased AQP2 mRNA expression by arginine vasopressin (AVP) but not by hypertonicity, which induces both TonEBP and NF-?B activity. AVP-induced AQP2 expression was similarly decreased by the flavoprotein inhibitor diphenyleneiodonium. siNOX4 altered neither TonEBP nor NF-?B activity but attenuated AVP-inducible cellular cAMP concentration, PKA activity and CREB phosphorylation as well as AQP2 mRNA expression induced by forskolin, a potent activator of adenylate cyclase. The repressive effect of siNOX4 on AVP-induced AQP2 mRNA expression was abolished by the non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and was significantly decreased by selective PDE antagonists cilostamide and rolipram, but not vinpocetine, which respectively target PDE3, PDE4 and PDE1. Thus, by inhibiting PDE3 and PDE4 activity NOX4-derived ROS may contribute to V2R-cAMP-PKA signaling and enhance AQP2 transcription. PMID:24466344

Feraille, Eric; Dizin, Eva; Roth, Isabelle; Derouette, Jean-Paul; Szanto, Ildiko; Martin, Pierre-Yves; de Seigneux, Sophie; Hasler, Udo

2014-01-01

171

ClC-3 deficiency prevents apoptosis induced by angiotensin II in endothelial progenitor cells via inhibition of NADPH oxidase.  

PubMed

Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization and re-endothelialization in response to tissue ischemia and endothelial injury. It is reported that the circulating EPCs number is decreased during hypertension. However, the detailed mechanism is still unclear. Our previous studies have shown that ClC-3 chloride channel is up-regulated with the development of hypertension. This study aims to test whether ClC-3 participates in EPC apoptosis under the condition of increased oxidative stress in angiotensin II (Ang II)-induced hypertension. The results showed that stimulation with 10(-6)mol/L Ang II significantly up-regulated the endogenous ClC-3 expression and increased intracellular reactive oxygen species (ROS) generation in EPCs of wild type mice, accompanied by an enhanced NADPH oxidase activity and the expression of gp91(phox) (NOX-2), a key catalytic subunit of NADPH oxidase. However, these effects of Ang II were significantly reduced in EPCs of ClC-3(-/-) mice. Compared with control, treatment with Ang II induced EPCs apoptosis in wild type mice, concomitantly with declined Bcl-2/Bax ratio, depressed mitochondrial membrane potential and activation of poly(ADP-ribose) polymerase, which was remarkably prevented by both ClC-3 knockout and NADPH oxidase inhibitor apocynin. In addition, the role of ClC-3 deficiency in protecting EPCs against Ang II-induced oxidative stress and apoptosis was further confirmed in Ang II-infused hypertensive mice in vivo. In conclusion, ClC-3 deficiency inhibited Ang II-induced EPC apoptosis via suppressing ROS generation derived from NADPH oxidase. PMID:23873092

Liu, Jing; Zhang, Fei-Fei; Li, Lei; Yang, Jing; Liu, Jie; Guan, Yong-Yuan; Du, Yan-Hua

2013-10-01

172

The signaling pathway of NADPH oxidase and its role in glomerular diseases.  

PubMed

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), a major source of reactive oxygen species, is a critical mediator of redox signaling. It is well-documented that oxidative stress is associated with the development of glomerular diseases (GN). Hence, the Nox was also thought to be involved in the pathogenesis of GN. However, the expression of Nox in various GN was not consistent, the mechanisms by which the activity of the Nox enzymes in regulating renal cells remains unclear. Signaling pathways might be very important in the pathogenesis of GN. We performed this review to provide a relatively complete signaling pathways flowchart for Nox to the investigators who were interested in the role of Nox in the pathogenesis of GN. Here, we reviewed the signal transduction pathway of Nox and its role in the pathogenesis of GN. PMID:24156279

Mao, Song; Huang, Songming

2014-02-01

173

Rab27a regulates phagosomal pH and NADPH oxidase recruitment to dendritic cell phagosomes.  

PubMed

To prevent excessive degradation of internalized antigens, which could destroy the peptides recognized by T lymphocytes, dendritic cells have developed several strategies that limit proteolytic activity in phagosomes. The recruitment of the NADPH oxidase NOX2 prevents acidification of phagosomes, limiting antigen degradation. Here, we show that dendritic cells derived from Rab27a-deficient ashen mice show increased phagosome acidification and antigen degradation, causing a defect in antigen cross-presentation. Enhanced acidification results from a delay in the recruitment to phagosomes of a subset of lysosome-related organelles containing the membrane subunits of NOX2. The Rab27a-dependent recruitment of these "inhibitory lysosome-related organelles" to phagosomes continuously limits acidification and degradation of ingested particles in dendritic cells, thus promoting antigen cross-presentation. PMID:17351642

Jancic, Carolina; Savina, Ariel; Wasmeier, Christina; Tolmachova, Tanya; El-Benna, Jamel; Dang, Pham My-Chan; Pascolo, Steve; Gougerot-Pocidalo, Maire-Anne; Raposo, Graça; Seabra, Miguel C; Amigorena, Sebastian

2007-04-01

174

Deficiency of NADPH Oxidase Components p47phox and gp91phox Caused Granulomatous Synovitis and Increased Connective Tissue Destruction in Experimental Arthritis Models  

PubMed Central

Recent studies indicated that the nicotinamide dinucleotide phosphate oxidase (NADPH) oxidase-derived oxygen radicals plays a deleterious role in arthritis. To study this in more detail, gonarthritis was induced in NADPH oxidase-deficient mice. Mice received an intraarticular injection of either zymosan, to elicit an irritant-induced inflammation, or poly-l-lysine coupled lysozyme, to evoke an immune-complex mediated inflammation in passively immunized mice. In contrast to wild-type mice, arthritis elicited in both p47phox?/? and gp91?/? mice showed more severe joint inflammation, which developed into a granulomatous synovitis. Treatment with either Zileuton or cobra venom factor showed that the chemokines LTB4 and complement C3 were not the driving force behind the aggravated inflammation in these mice. Arthritic NADPH oxidase-deficient mice showed irreversible cartilage damage as judged by the enhanced aggrecan VDIPEN expression, and chondrocyte death. Furthermore, only in the absence of NADPH oxidase-derived oxygen radicals, the arthritic joints showed osteoclast-like cells, tartrate-resistant acid phosphatase (TRAP)-positive/multinucleated cells, extensive bone erosion, and osteolysis. The enhanced synovial gene expression of tumor necrosis factor-?, interleukin-1?, matrix metalloproteinase (MMP)-3, MMP-9 and receptor activator of NF-?B ligand (RANKL) might contribute to the aggravated arthritis in the NADPH oxidase-deficient mice. This showed that the involvement of NADPH oxidase in arthritis is probably far more complex and that oxygen radicals might also be important in controlling disease severity, and reducing joint inflammation and connective tissue damage. PMID:14507659

van de Loo, Fons A. J.; Bennink, Miranda B.; Arntz, Onno J.; Smeets, Ruben L.; Lubberts, Erik; Joosten, Leo A. B.; van Lent, Peter L. E. M.; Coenen-de Roo, Christina J. J.; Cuzzocrea, Salvatore; Segal, Brahm H.; Holland, Steven M.; van den Berg, Wim B.

2003-01-01

175

NADPH Oxidase and Angiogenesis Following Endothelin-1 Induced Stroke in Rats: Role for Nox2 in Brain Repair  

PubMed Central

NADPH oxidases contribute to brain injury, yet they may also have a role in brain repair, particularly in vascular signaling and angiogenesis. This study determined the temporal and spatial profile of NADPH oxidase subunit expression/activity concurrently with angiogenesis in the brain following transient ischemic stroke induced by prolonged constriction of the middle cerebral artery by perivascular injection of endothelin-1 in conscious Hooded Wistar rats (n = 47). VEGF mRNA expression was increased in the ipsilateral cortex and striatum between 6 h and 28 days post-stroke concurrently with a marked increase in Nox2 mRNA expression up to 7 days, and increased Nox4 mRNA expression detected between 7 and 28 days. Point counting of blood vessels using Metamorph imaging software showed increased vascular sprouting between 3 and 7 days after stroke with new vascular networks detected in the core infarct region by 14 days. Angiogenic blood vessels 3 and 7 days post-stroke were observed to co-localise with both Nox2 antibody and dihydroethidium fluorescence suggesting a role for Nox2 generated superoxide during the phase of vascular remodeling, whilst Nox4 expression was detected once new cerebral vessels had formed. These results indicate for the first time that ROS signaling through a cerebrovascular Nox2 NADPH oxidase may be important in initiating brain angiogenesis. PMID:24961316

Taylor, Caroline J.; Weston, Robert M.; Dusting, Gregory J.; Roulston, Carli L.

2013-01-01

176

Apocynin inhibits NADPH oxidase in phagocytes but stimulates ROS production in non-phagocytic cells  

Microsoft Academic Search

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH oxidase. Since it acts as a potent inhibitor in studies with neutrophils and macrophages, no inhibitory effect can often be found in non-phagocyte cells. In our experiments, apocynin even stimulated reactive oxygen species (ROS) production by vascular fibroblasts. Even when added to macrophages, apocynin initially caused

Martin Vejražka; Radan Mí?ek; Stanislav Štípek

2005-01-01

177

Oxidative stress in hemodialysis patients: Is NADPH oxidase complex the culprit?  

Microsoft Academic Search

Oxidative stress in hemodialysis patients: Is NADPH oxidase complex the culprit?Oxidative stress results from an imbalance between oxidant production, including reactive oxygen species (ROS), reactive nitrogen species (RNS), chlorinated compounds, and antioxidant defense mechanisms. Most reports prove that oxidative stress is present in ESRD patients. Several studies tend to accreditate the hypothesis by which oxidative stress is a strong co-factor

Marion Morena; Jean-Paul Cristol; Lynne Senécal; Helene Leray-Moragues; Detlef Krieter; Bernard Canaud

2002-01-01

178

Role of the NADPH Oxidases DUOX and NOX4 in Thyroid Oxidative Stress  

PubMed Central

Somatic mutations are present at high levels in the rat thyroid gland, indicating that the thyrocyte is under oxidative stress, a state in which cellular oxidant levels are high. The most important class of free radicals, or reactive metabolites, is reactive oxygen species (ROS), such as superoxide anion (O2-), hydroxyl radical (OH) and hydrogen peroxide (H2O2). The main source of ROS in every cell type seems to be mitochondrial respiration; however, recent data support the idea that NADPH:O(2) oxidoreductase flavoproteins or simply NADPH oxidases (NOX) are enzymes specialized in controlled ROS generation at the subcellular level. Several decades ago, high concentrations of H2O2 were detected at the apical surface of thyrocytes, where thyroid hormone biosynthesis takes place. Only in the last decade has the enzymatic source of H2O2 involved in thyroid hormone biosynthesis been well characterized. The cloning of two thyroid genes encoding NADPH oxidases dual oxidases 1 and 2 (DUOX1 and DUOX2) revealed that DUOX2 mutations lead to hereditary hypothyroidism in humans. Recent reports have also described the presence of NOX4 in the thyroid gland and have suggested a pathophysiological role of this member of the NOX family. In the present review, we describe the participation of NADPH oxidases not only in thyroid physiology but also in gland pathophysiology, particularly the involvement of these enzymes in the regulation of thyroid oxidative stress. PMID:24847449

Carvalho, Denise P.; Dupuy, Corinne

2013-01-01

179

NADPH Oxidases in Heart Failure: Poachers or Gamekeepers?  

PubMed Central

Abstract Significance: Oxidative stress is involved in the pathogenesis of heart failure but clinical antioxidant trials have been unsuccessful. This may be because effects of reactive oxygen species (ROS) depend upon their source, location, and concentration. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) proteins generate ROS in a highly regulated fashion and modulate several components of the heart failure phenotype. Recent Advances: Two Nox isoforms, Nox2 and Nox4, are expressed in the heart. Studies using gene-modified mice deficient in Nox2 activity indicate that Nox2 activation contributes to angiotensin II–induced cardiomyocyte hypertrophy, atrial fibrillation, and the development of interstitial fibrosis but may also positively modulate physiological excitation-contraction coupling. Nox2 contributes to myocyte death under stress situations and plays important roles in postmyocardial infarction remodeling, in part by modulating matrix metalloprotease activity. In contrast to Nox2, Nox4 is constitutively active at a low level and induces protective effects in the heart under chronic stress, for example, by maintaining myocardial capillary density. However, high levels of Nox4 could have detrimental effects. Critical Issues: The effects of Nox proteins during the development of heart failure likely depend upon the isoform, activation level, and cellular distribution, and may include beneficial as well as detrimental effects. More needs to be learnt about the precise regulation of abundance and biochemical activity of these proteins in the heart as well as the downstream signaling pathways that they regulate. Future Directions: The development of specific approaches to target individual Nox isoforms and/or specific cell types may be important for the achievement of therapeutic efficacy in heart failure. Antioxid. Redox Signal. 18, 1024–1041. PMID:22747566

Zhang, Min; Perino, Alessia; Ghigo, Alessandra; Hirsch, Emilio

2013-01-01

180

Bacillus Calmette-Guerin Infection in NADPH Oxidase Deficiency: Defective Mycobacterial Sequestration and Granuloma Formation  

PubMed Central

Patients with chronic granulomatous disease (CGD) lack generation of reactive oxygen species (ROS) through the phagocyte NADPH oxidase NOX2. CGD is an immune deficiency that leads to frequent infections with certain pathogens; this is well documented for S. aureus and A. fumigatus, but less clear for mycobacteria. We therefore performed an extensive literature search which yielded 297 cases of CGD patients with mycobacterial infections; M. bovis BCG was most commonly described (74%). The relationship between NOX2 deficiency and BCG infection however has never been studied in a mouse model. We therefore investigated BCG infection in three different mouse models of CGD: Ncf1 mutants in two different genetic backgrounds and Cybb knock-out mice. In addition, we investigated a macrophage-specific rescue (transgenic expression of Ncf1 under the control of the CD68 promoter). Wild-type mice did not develop severe disease upon BCG injection. In contrast, all three types of CGD mice were highly susceptible to BCG, as witnessed by a severe weight loss, development of hemorrhagic pneumonia, and a high mortality (?50%). Rescue of NOX2 activity in macrophages restored BCG resistance, similar as seen in wild-type mice. Granulomas from mycobacteria-infected wild-type mice generated ROS, while granulomas from CGD mice did not. Bacterial load in CGD mice was only moderately increased, suggesting that it was not crucial for the observed phenotype. CGD mice responded with massively enhanced cytokine release (TNF-?, IFN-?, IL-17 and IL-12) early after BCG infection, which might account for severity of the disease. Finally, in wild-type mice, macrophages formed clusters and restricted mycobacteria to granulomas, while macrophages and mycobacteria were diffusely distributed in lung tissue from CGD mice. Our results demonstrate that lack of the NADPH oxidase leads to a markedly increased severity of BCG infection through mechanisms including increased cytokine production and impaired granuloma formation. PMID:25188296

Deffert, Christine; Schappi, Michela G.; Pache, Jean-Claude; Cachat, Julien; Vesin, Dominique; Bisig, Ruth; Ma Mulone, Xiaojuan; Kelkka, Tiina; Holmdahl, Rikard

2014-01-01

181

The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress  

SciTech Connect

We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-{kappa}B and decreased intracellular level of its inhibitor IkB{alpha}. These effects, accompanied by increased production of H{sub 2}O{sub 2}, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-{kappa}B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.

Riganti, Chiara [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Torino (Italy)], E-mail: chiara.riganti@unito.it; Costamagna, Costanzo; Doublier, Sophie; Miraglia, Erica; Polimeni, Manuela; Bosia, Amalia; Ghigo, Dario [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Torino (Italy)

2008-05-01

182

Increased NAD(P)H oxidase-mediated superoxide production in renovascular hypertension: Evidence for an involvement of protein kinase C  

Microsoft Academic Search

Increased NAD(P)H oxidase-mediated superoxide production in renovascular hypertension: Evidence for an involvement of protein kinase C.BackgroundAngiotensin II infusion has been shown to cause hypertension and endothelial dysfunction and to increase superoxide (O?·2) production in vascular tissue, mainly via an activation of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-dependent oxidase, the most significant O?·2 source in endothelial and\\/or smooth muscle cells. With these

THOMAS HEITZER; ULRICH WENZEL; ULRICH HINK; DIRK KROLLNER; MIKHAIL SKATCHKOV; ROLF A K STAHL; ROLAND MACHARZINA; JAN H BRÄSEN; THOMAS MEINERTZ; THOMAS MÜNZEL

1999-01-01

183

Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress  

NASA Technical Reports Server (NTRS)

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

McNally, J. Scott; Davis, Michael E.; Giddens, Don P.; Saha, Aniket; Hwang, Jinah; Dikalov, Sergey; Jo, Hanjoong; Harrison, David G.

2003-01-01

184

Hu-Lu-Ba-Wan Attenuates Diabetic Nephropathy in Type 2 Diabetic Rats through PKC-?/NADPH Oxidase Signaling Pathway  

PubMed Central

Hu-Lu-Ba-Wan (HLBW) is a Chinese herbal prescription used to treat kidney deficiency. The aim of this study was to explore the effect and mechanism of HLBW on diabetic nephropathy (DN) in type 2 diabetic rats. The rat model of DN was established by being fed a high-fat diet and intravenous injection of streptozotocin. Then, HLBW decoction was administered for 16 weeks. Blood glucose level, lipid profile, renal function, 24-hour total urinary protein, and albumin content were examined. Renal morphology and superoxide anion levels were evaluated. The activity of nicotinamide-adenine dinucleotide phosphate (NADPH) and protein kinase C-alpha (PKC-?) related genes expression in renal tissue were also determined. Our data demonstrated that HLBW significantly improved hyperglycemia, hyperlipidemia, and proteinuria in diabetic rats compared with those of control group. HLBW also alleviated glomerular expansion and fibrosis, extracellular matrix accumulation and effacement of the foot processes. Additionally, HLBW reduced superoxide anion level, NADPH oxidase activity, the protein and mRNA expressions of p47phox, and the protein expression of phosphorylated PKC-? in renal tissue. These results suggest that HLBW is effective in the treatment of DN in rats. The underlying mechanism may be related to the attenuation of renal oxidative stress via PKC-?/NADPH oxidase signaling pathway. PMID:23878600

Zhou, Lishan; Dong, Hui; Huang, Yi; Xu, Lijun; Zou, Xin; Wang, Kaifu; Chen, Guang; Lu, Fuer

2013-01-01

185

Redefining the major contributors to superoxide production in contracting skeletal muscle. The role of NAD(P)H oxidases.  

PubMed

The production of reactive oxygen and nitrogen species (RONS) by skeletal muscle is important as it (i) underlies oxidative damage in many degenerative muscle pathologies and (ii) plays multiple regulatory roles by fulfilling important cellular functions. Superoxide and nitric oxide (NO) are the primary radical species produced by skeletal muscle and studies in the early 1980s demonstrated that their generation is augmented during contractile activity. Over the past 30 years considerable research has been undertaken to identify the major sites that contribute to the increased rate of RONS generation in response to contractions. It is widely accepted that NO is regulated by the nitric oxide synthases, however the sites that modulate changes in superoxide during exercise remain unclear. Despite the initial indications that the mitochondrial electron transport chain was the predominant source of superoxide during activity, with the development of analytical methods a number of alternative potential sites have been identified including the NAD(P)H oxidases, xanthine oxidase, cyclooxygenases, and lipoxygenases linked to the activity of the phospholipase A2 enzymes. In the present review we outline the subcellular sites that modulate intracellular changes in superoxide in skeletal muscle and based on the available experimental evidence in the literature we conclude that the NAD(P)H oxidases are likely to be the major superoxide generating sources in contracting skeletal muscle. PMID:23915064

Sakellariou, G K; Jackson, M J; Vasilaki, A

2014-01-01

186

NADPH oxidase-2 is a key regulator of human dermal fibroblasts: a potential therapeutic strategy for the treatment of skin fibrosis.  

PubMed

The proliferation of human skin dermal fibroblasts (HDFs) is a critical step in skin fibrosis, and transforming growth factor-beta1 (TGF-?1) exerts pro-oxidant and fibrogenic effects on HDFs. In addition, the oxidative stress system has been implicated in the pathogenesis of skin disease. However, the role of NADPH oxidase as a mediator of TGF-?1-induced effects in HDFs remains unknown. Thus, our aim was to investigate the role of NADPH in human skin dermal fibroblasts. Primary fibroblasts were cultured and pretreated with various stimulants. Real-time Q-PCR and Western blotting analyses were used for mRNA and protein detection. In addition, siRNA technology was applied for gene knock-down analysis. Hydrogen peroxide production and 2',7'-dichlorofluorescein diacetate (DCFDA) measurement assay were performed. Here, our findings demonstrated that HDFs express key components of non-phagocytic NADPH oxidase mRNA. TGF-?1 induced NOX2 and reactive oxygen species formation via NADPH oxidase activity. In contrast, NOX3 was barely detectable, and other NOXs did not display significant changes. In addition, TGF-?1 phosphorylated MAPKs and increased activator protein-1 (AP-1) in a redox-sensitive manner, and NOX2 suppression inhibited baseline and TGF-?1-mediated stimulation of Smad2 phosphorylation. Moreover, TGF-?1 stimulated cell proliferation, migration, collagen I and fibronectin expression, and bFGF and PAI-1 secretion: these effects were attenuated by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and NOX2 siRNA. Importantly, NOX2 siRNA suppresses collagen production in primary keloid dermal fibroblasts. These findings provide the proof of concept for NADPH oxidase as a potential target for the treatment of skin fibrosis. PMID:24981855

Zhang, Guo-You; Wu, Liang-Cai; Dai, Tao; Chen, Shi-Yi; Wang, An-Yuan; Lin, Kang; Lin, Da-Mu; Yang, Jing-Quan; Cheng, Biao; Zhang, Li; Gao, Wei-Yang; Li, Zhi-Jie

2014-09-01

187

Oral treatment with the NADPH oxidase antagonist apocynin mitigates clinical and pathological features of parkinsonism in the MPTP marmoset model.  

PubMed

This study evaluates the therapeutic efficacy of the NADPH oxidase inhibitor apocynin, isolated as principal bioactive component from the medicinal plant Picrorhiza kurroa, in a marmoset MPTP model of Parkinson's disease (PD). The methoxy-substituted catechol apocynin has a similar structure as homovanillic acid (HVA), a metabolite of dopamine (DA). Apocynin acquires its selective inhibitory capacity of the reactive oxygen species generating NADPH oxidase via metabolic activation by myeloperoxidase (MPO). As MPO is upregulated in activated brain microglia cells of PD patients and in MPTP animal models, the conditions for metabolic activation of apocynin and inhibition of microglia NADPH oxidase are in place. Marmoset monkeys received oral apocynin (100 mg/kg; p.o.) (n?=?5) or Gum Arabica (controls; n?=?5) three times daily until the end of the study, starting 1 week before PD induction with MPTP (1 mg/kg?s.c. for 8 days). Parkinsonian symptoms, motor function, home-cage activity and body weight were monitored to assess the disease development and severity. Post-mortem numbers of the tyrosine hydroxylase expressing DA neurons in the substantia nigra were counted. During the MPTP injections, apocynin limited the body weight loss and relieved parkinsonian symptoms compared to controls (Linear regression, P?activity with 32 % (P?=?0.029), indicating anti-Parkinson efficacy. Apocynin also increased the number of surviving DA neurons in MPTP-treated marmosets with 8.5 % (P?=?0.059), indicating a tendency towards a neuroprotective efficacy. In conclusion, compensation for the loss of DA and its metabolite HVA by apocynin mitigates the PD progression and limits the parkinsonian signs and motor-function deterioration. PMID:23504289

Philippens, Ingrid H C H M; Wubben, Jacqueline A; Finsen, Bente; 't Hart, Bert A

2013-06-01

188

NADPH oxidase-dependent redox signaling in TGF-?-mediated fibrotic responses?  

PubMed Central

Uncontrolled fibrosis in organs like heart, kidney, liver and lung is detrimental and may lead to end-stage organ failure. Currently there is no effective treatment for fibrotic disorders. Transforming growth factor (TGF)-? has a fundamental role in orchestrating the process of fibrogenesis; however, interventions directly targeting TGF-? would have undesired systemic side effects due to the multiple physiological functions of TGF-?. Further characterization of the downstream signaling pathway(s) involved in TGF-?-mediated fibrosis may lead to discovery of novel treatment strategies for fibrotic disorders. Accumulating evidence suggests that Nox4 NADPH oxidase may be an important downstream effector in mediating TGF-?-induced fibrosis, while NADPH oxidase-dependent redox signaling may in turn regulate TGF-?/Smad signaling in a feed-forward manner. It is proposed that pharmacological inhibition of the Nox4 function may represent a novel approach in treatment of fibrotic disorders. PMID:24494202

Jiang, Fan; Liu, Guei-Sheung; Dusting, Gregory J.; Chan, Elsa C.

2014-01-01

189

Elevated systemic TGF-beta impairs aortic vasomotor function through activation of NADPH oxidase-driven superoxide production and leads to hypertension, myocardial remodeling, and increased plaque formation in apoE(-/-) mice.  

PubMed

The role of circulating, systemic TGF-beta levels in endothelial function is not clear. TGF-beta(1) may cause endothelial dysfunction in apolipoprotein E-deficient (apoE(-/-)) mice via stimulation of reactive oxygen species (ROS) production by the NADPH oxidase (NOX) system and aggravate aortic and heart remodeling and hypertension. Thoracic aorta (TA) were isolated from 4-mo-old control (C57Bl/6), apoE(-/-), TGF-beta(1)-overexpressing (TGFbeta(1)), and crossbred apoE(-/-) x TGFbeta(1) mice. Endothelium-dependent relaxation was measured before and after incubation with apocynin (NOX inhibitor) or superoxide dismutase (SOD; ROS scavenger). Superoxide production within the vessel wall was determined by dihydroethidine staining under confocal microscope. In 8-mo-old mice, aortic and myocardial morphometric changes, plaque formation by en face fat staining, and blood pressure were determined. Serum TGF-beta(1) levels (ELISA) were elevated in TGFbeta(1) mice without downregulation of TGF-beta-I receptor (immunohistochemistry). In the aortic wall, superoxide production was enhanced and NO-dependent relaxation diminished in apoE(-/-) x TGFbeta(1) mice but improved significantly after apocynin or SOD. Myocardial capillary density was reduced, fibrocyte density increased, aortic wall was thicker, combined lesion area was greater, and blood pressure was higher in the apoE(-/-) x TGFbeta vs. C57Bl/6 mice. Our results demonstrate that elevated circulating TGF-beta(1) causes endothelial dysfunction through NOX activation-induced oxidative stress, accelerating atherosclerosis and hypertension in apoE(-/-) mice. These findings may provide a mechanism explaining accelerated atherosclerosis in patients with elevated plasma TGFbeta(1). PMID:20511416

Buday, Anna; Orsy, Petra; Godó, Mária; Mózes, Miklós; Kökény, Gábor; Lacza, Zsombor; Koller, Akos; Ungvári, Zoltán; Gross, Marie-Luise; Benyó, Zoltán; Hamar, Péter

2010-08-01

190

NADPH oxidases in cardiovascular disease: insights from in vivo models and clinical studies  

Microsoft Academic Search

NADPH oxidase family enzymes (or NOXs) are the major sources of reactive oxygen species (ROS) that are implicated in the pathophysiology\\u000a of many cardiovascular diseases. These enzymes appear to be especially important in the modulation of redox-sensitive signalling\\u000a pathways that underlie key cellular functions such as growth, differentiation, migration and proliferation. Seven distinct\\u000a members of the family have been identified

Alexander Sirker; Min Zhang; Ajay M. Shah

191

NADPH Oxidase Contributes to Angiotensin II Signaling in the Nucleus Tractus Solitarius  

Microsoft Academic Search

Angiotensin II (AngII), acting through angiotensin type 1 (AT1 ) receptors, exerts powerful effects on central autonomic networks regulating cardiovascular homeostasis and fluid balance; however, the mechanisms of AngII signaling in functionally defined central autonomic neurons have not been fully elucidated. In vascular cells, reactive oxygen species (ROS) generated by the enzyme NADPH oxidase play a major role in AngII

Gang Wang; Josef Anrather; Jie Huang; Robert C. Speth; Virginia M. Pickel; Costantino Iadecola

2004-01-01

192

The NADPH-oxidase AtrbohB plays a role in Arabidopsis seed after-ripening  

Microsoft Academic Search

Summary • Seeds can enter a state of dormancy, in which they do not germinate under optimal environmental conditions. Dormancy can be broken during seed after-rip- ening in the low-hydrated state. • By screening enhancer trap lines of Arabidopsis, we identified a role for the NADPH-oxidase AtrbohB in after-ripening. Semiquantitative PCR was used to investigate AtrbohB transcripts in seeds. These

Anna Catharina Carstens; Ada Linkies; Miguel Angel Torres; Gerhard Leubner-Metzger

2009-01-01

193

METHAMPHETAMINE ALTERS OCCLUDIN EXPRESSION VIA NADPH OXIDASE-INDUCED OXIDATIVE INSULT AND INTACT CAVEOLAE  

PubMed Central

Methamphetamine (METH) is a drug of abuse with neurotoxic and vascular effects that may be mediated by reactive oxygen species (ROS). However, potential sources of METH-induced generation of ROS are not fully understood. The present study is focused on the role of NAD(P)H oxidase (NOX) in METH-induced dysfunction of brain endothelial cells. Treatment with METH induced a time-dependent increase in phosphorylation of NOX subunit p47, followed by its binding with gp91 and p22, and the formation of an active NOX complex. An increase in NOX activity was associated with elevated production of ROS, alterations of occludin levels, and increased transendothelial migration of monocytes. Inhibition of NOX by NSC 23766 attenuated METH-induced ROS generation, changes in occludin protein levels, and monocyte migration. Because an active NOX complex is localized to caveolae, we next evaluated the role of caveolae in METH-mediated toxicity to brain endothelial cells. Treatment with METH induced phosphorylation of ERK1/2 and caveolin-1 protein. Inhibition of ERK1/2 activity or caveolin-1 silencing protected against METH-induced alterations of occludin levels. These findings indicate an important role of NOX and functional caveolae in METH-induced oxidative stress in brain endothelial cells that contribute to the subsequent alterations of occludin levels and transendothelial migration of inflammatory cells. PMID:21435178

Park, Minseon; Hennig, Bernhard; Toborek, Michal

2011-01-01

194

Oxidative Stress in Atrial Fibrillation: An Emerging Role of NADPH Oxidase  

PubMed Central

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Patients with AF have up to seven-fold higher risk of suffering from ischemic stroke. Better understanding of etiologies of AF and its thromboembolic complications are required for improved patient care, as current anti-arrhythmic therapies have limited efficacy and off target effects. Accumulating evidence has implicated a potential role of oxidative stress in the pathogenesis of AF. Excessive production of reactive oxygen species (ROS) is likely involved in the structural and electrical remodeling of the heart, contributing to fibrosis and thrombosis. In particular, NADPH oxidase (NOX) has emerged as a potential enzymatic source for ROS production in AF based on growing evidence from clinical and animal studies. Indeed, NOX can be activated by known upstream triggers of AF such as angiotensin II and atrial stretch. In addition, treatments such as Statins, antioxidants, ACEI or AT1RB have been shown to prevent post-operative AF; among which ACEI/AT1RB and Statins can attenuate NOX activity. On the other hand, detailed molecular mechanisms by which specific NOX isoform(s) are involved in the pathogenesis of AF and the extent to which activation of NOX plays a causal role in AF development remains to be determined. The current review discusses causes and consequences of oxidative stress in AF with a special focus on the emerging role of NOX pathways. PMID:23643589

Youn, Ji-Youn; Zhang, Jun; Zhang, Yixuan; Chen, Houzao; Liu, Depei; Ping, Peipei; Weiss, James N.; Cai, Hua

2013-01-01

195

Anti-inflammatory effects of apocynin, an inhibitor of NADPH oxidase, in airway inflammation.  

PubMed

Oxidative stress is implicated in the pathogenesis of allergic asthma and remains an attractive target for the prevention of the disease. Herein, we investigated the anti-inflammatory effects of apocynin, a NADPH oxidase (NOX) inhibitor, in both in vitro and in vivo allergen-induced experimental asthma mediated by Th2 hyperresponsiveness. Apocynin showed potential antioxidant activities and inhibitory effects on the activation of redox-sensitive transcription factors, such as NF-?B and AP-1, induced by pro-inflammatory stimuli, such as TNF-?, lipopolysaccharide and Poly I:C, and that inhibited the production of pro-inflammatory cytokines, such as TNF-?, IL-1? and IL-6. In in vivo experimental asthma model, moreover, apocynin significantly attenuated ovalbumin-induced airway hyperresponsiveness and inflammation, as shown by the attenuation of total inflammatory cell and soluble product influx into bronchoalveolar lavage fluid, such as macrophages, eosinophils, IL-4, IL-5, IL-12, IL-13 and TNF-?. Apocynin also significantly reduced lung inflammation in the tissues. Altogether, these results suggest that apocynin may be useful in the treatment of inflammatory diseases induced by oxidative stress through NOX activity. PMID:21709687

Kim, So Yong; Moon, Keun-Ai; Jo, Hee-Yeon; Jeong, Sookyung; Seon, Seung-Han; Jung, Eunyu; Cho, You Sook; Chun, Eunyoung; Lee, Ki-Young

2012-04-01

196

NADPH Oxidase and the Degeneration of Dopaminergic Neurons in Parkinsonian Mice  

PubMed Central

Several lines of investigation have implicated oxidative stress in Parkinson's disease (PD) pathogenesis, but the mechanisms involved are still unclear. In this study, we characterized the involvement of NADPH oxidase (Nox), a multisubunit enzyme that catalyzes the reduction of oxygen, in the 6-hydroxydopamine- (6-OHDA-) induced PD mice model and compared for the first time the effects of this neurotoxin in mice lacking gp91phox?/?, the catalytic subunit of Nox2, and pharmacological inhibition of Nox with apocynin. Six-OHDA induced increased protein expression of p47phox, a Nox subunit, in striatum. gp91phox?/? mice appear to be completely protected from dopaminergic cell loss, whereas the apocynin treatment conferred only a limited neuroprotection. Wt mice treated with apocynin and gp91phox?/? mice both exhibited ameliorated apomorphine-induced rotational behavior. The microglial activation observed within the striatum and the substantia nigra pars compacta (SNpc) of 6-OHDA-injected Wt mice was prevented by apocynin treatment and was not detected in gp91phox?/? mice. Apocynin was not able to attenuate astrocyte activation in SN. The results support a role for Nox2 in the 6-OHDA-induced degeneration of dopaminergic neurons and glial cell activation in the nigrostriatal pathway and reveal that no comparable 6-OHDA effects were observed between apocynin-treated and gp91phox?/? mice groups. PMID:24379900

Hernandes, Marina S.; Cafe-Mendes, Cecilia C.; Britto, Luiz R. G.

2013-01-01

197

NADPH oxidase inhibition ameliorates Trypanosoma cruzi-induced myocarditis during Chagas disease.  

PubMed

Trypanosoma cruzi, the aetiological agent of Chagas disease, invades nucleated mammalian cells including macrophages. In this study, we investigated the crosstalk between T. cruzi-induced immune activation of reactive oxygen species (ROS) and pro-inflammatory responses, and their role in myocardial pathology. Splenocytes of infected mice (C3H/HeN) responded to Tc-antigenic stimulus by more than a two-fold increase in NADPH oxidase (NOX) activity, ROS generation, cytokine production (IFN-? > IL-4 > TNF? > IL1-?? IL6), and predominant expansion of CD4(+) and CD8(+) T cells. Inhibition of NOX, but not of myeloperoxidase and xanthine oxidase, controlled the ROS (>98%) and cytokine (70-89%) release by Tc-stimulated splenocytes of infected mice. Treatment of infected mice with apocynin (NOX inhibitor) in drinking water resulted in a 50-90% decline in endogenous NOX/ROS and cytokine levels, and splenic phagocytes' proliferation. The splenic percentage of T cells was maintained, though more than a 40% decline in splenic index (spleen weight/body weight) indicated decreased T-cell proliferation in apocynin-treated/infected mice. The blood and tissue parasite burden were significantly increased in apocynin-treated/infected mice, yet acute myocarditis, ie inflammatory infiltrate consisting of macrophages, neutrophils, and CD8(+) T cells, and tissue oxidative adducts (eg 8-isoprostanes, 3-nitrotyrosine, and 4-hydroxynonenal) were diminished in apocynin-treated/infected mice. Consequently, hypertrophy (increased cardiomyocytes' size and ?-MHC, BNP, and ANP mRNA levels) and fibrosis (increased collagen, glycosaminoglycans, and lipid contents) of the heart during the chronic phase were controlled in apocynin-treated mice. We conclude that NOX/ROS is a critical regulator of the splenic response (phagocytes, T cells, and cytokines) to T. cruzi infection, and bystander effects of heart-infiltrating phagocytes and CD8(+) T cells resulting in cardiac remodelling in chagasic mice. PMID:21952987

Dhiman, Monisha; Garg, Nisha Jain

2011-12-01

198

WY-14,643 induced cell proliferation and oxidative stress in mouse liver are independent of NADPH oxidase.  

PubMed

Long-term exposure of rodents to peroxisome proliferators leads to increases in peroxisomes, hepatocellular proliferation, oxidative damage, suppressed apoptosis, and ultimately results in the development of hepatic adenomas and carcinomas. Peroxisome proliferators-activated receptor (PPAR)alpha was shown to be required for these pleiotropic responses; however, Kupffer cells, resident liver macrophages, were also identified as playing a role in peroxisome proliferators-induced effects, independently of PPARalpha. Previous studies showed that oxidants from NADPH (nicotinamide adenine dinucleotide phosphate, reduced) oxidase mediate acute effects of peroxisome proliferators in rodent liver. To determine if Kupffer cell oxidants are also involved in chronic effects, NADPH oxidase-deficient (p47(phox)-null) mice were fed 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (WY-14,643)-containing diet (0.1% wt/wt) for 1 week, 5 weeks, or 5 months along with Pparalpha-null and wild type mice. As expected, no change in liver size, cell replication rates, or other phenotypic effects of peroxisome proliferators were observed in Pparalpha-null mice. Through 5 months of treatment, the p47(phox)-null and wild type mice exhibited peroxisome proliferators-induced adverse liver effects, along with increased oxidative DNA damage and increased cell proliferation, a response that is potentially mediated through nuclear factor kappa B (NFkB). Suppressed apoptosis caused by WY-14,643 was dependent on both NADPH oxidase and PPARalpha. Collectively, these findings suggest that involvement of Kupffer cells in WY-14,643-induced parenchymal cell proliferation and oxidative stress in rodent liver is an acute phenomenon that is not relevant to long-term exposure, but they are still involved in chronic apoptotic responses. These results provide new insight for understanding the mode of hepatocarcinogenic action of peroxisome proliferators. PMID:17483499

Woods, Courtney G; Burns, Amanda M; Bradford, Blair U; Ross, Pamela K; Kosyk, Oksana; Swenberg, James A; Cunningham, Michael L; Rusyn, Ivan

2007-08-01

199

Hydrogen peroxide generated by NADPH oxidase is involved in high blue-light-induced chloroplast avoidance movements in Arabidopsis  

NASA Astrophysics Data System (ADS)

One of the most important functions of blue light is to induce chloroplast movements by reducing the damage to photosynthetic machinery under excess light. Hydrogen peroxide (H2O2), generated by various environmental stimuli, can act as a signaling molecule that regulates a number of developmental processes and environmental responses. To investigate whether H2O2 is involved in high blue light-induced chloroplast avoidance movements, we use luminescence spectrometer to observe H2O2 generation with the assistance of the fluorescence probe dichlorofluorescin diacetate (H2DCF-DA). After treatment with high blue light, a large quantity of H2O2 indicated by the fluorescence intensity of DCF is produced in a dose-dependent manner in leaf strip of Arabidopsis. Enzymatic assay shows that the activity of NADPH oxidase, which is a major site for H2O2 generation, also rapidly increases in treated strips. Exogenously applied H2O2 can promote the high blue light-induced chloroplast movements. Moreover, high blue light-induced H2O2 generation can be abolished completely by addition of exogenous catalase (CAT), and partly by diphenylene iodonium (DPI) and dichlorophenyl dimethylurea (DCMU), which are an NADPH oxidase inhibitor and a blocker of electron transport chain. And subsequent chloroplast movements can be abolished by CAT and DPI, but not by DCMU. These results presented here suggested that high blue light can induce oxidative burst, and NADPH oxidase as a major producer for H2O2 is involved in blue light-induced chloroplast avoidance movements.

Wen, Feng; Xing, Da; Zhang, Lingrui

2009-08-01

200

Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation.  

PubMed

Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-?B activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia. PMID:24326071

Arai, Yasuyuki; Nishinaka, Yoko; Arai, Toshiyuki; Morita, Makiko; Mizugishi, Kiyomi; Adachi, Souichi; Takaori-Kondo, Akifumi; Watanabe, Tomohiro; Yamashita, Kouhei

2014-01-10

201

Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth.  

PubMed

Tip-localized reactive oxygen species (ROS) were detected in growing pollen tubes by chloromethyl dichlorodihydrofluorescein diacetate oxidation, while tip-localized extracellular superoxide production was detected by nitroblue tetrazolium (NBT) reduction. To investigate the origin of the ROS we cloned a fragment of pollen specific tobacco NADPH oxidase (NOX) closely related to a pollen specific NOX from Arabidopsis. Transfection of tobacco pollen tubes with NOX-specific antisense oligodeoxynucleotides (ODNs) resulted in decreased amount of NtNOX mRNA, lower NOX activity and pollen tube growth inhibition. The ROS scavengers and the NOX inhibitor diphenylene iodonium chloride (DPI) inhibited growth and ROS formation in tobacco pollen tube cultures. Exogenous hydrogen peroxide (H2O2) rescued the growth inhibition caused by NOX antisense ODNs. Exogenous CaCl2 increased NBT reduction at the pollen tube tip, suggesting that Ca2+ increases the activity of pollen NOX in vivo. The results show that tip-localized ROS produced by a NOX enzyme is needed to sustain the normal rate of pollen tube growth and that this is likely to be a general mechanism in the control of tip growth of polarized plant cells. PMID:17504458

Potocký, Martin; Jones, Mark A; Bezvoda, Radek; Smirnoff, Nicholas; Zárský, Viktor

2007-01-01

202

JNK and NADPH oxidase involved in fluoride-induced oxidative stress in BV-2 microglia cells.  

PubMed

Excessive fluoride may cause central nervous system (CNS) dysfunction, and oxidative stress is a recognized mode of action of fluoride toxicity. In CNS, activated microglial cells can release more reactive oxygen species (ROS), and NADPH oxidase (NOX) is the major enzyme for the production of extracellular superoxide in microglia. ROS have been characterized as an important secondary messenger and modulator for various mammalian intracellular signaling pathways, including the MAPK pathways. In this study we examined ROS production and TNF- ? , IL-1 ? inflammatory cytokines releasing, and the expression of MAPKs in BV-2 microglia cells treated with fluoride. We found that fluoride increased JNK phosphorylation level of BV-2 cells and pretreatment with JNK inhibitor SP600125 markedly reduced the levels of intracellular O?(·-) and NO. NOX inhibitor apocynin and iNOS inhibitor SMT dramatically decreased NaF-induced ROS and NO generations, respectively. Antioxidant melatonin (MEL) resulted in a reduction in JNK phosphorylation in fluoride-stimulated BV-2 microglia. The results confirmed that NOX and iNOS played an important role in fluoride inducing oxidative stress and NO production and JNK took part in the oxidative stress induced by fluoride and meanwhile also could be activated by ROS in fluoride-treated BV-2 cells. PMID:24072958

Yan, Ling; Liu, Shengnan; Wang, Chen; Wang, Fei; Song, Yingli; Yan, Nan; Xi, Shuhua; Liu, Ziyou; Sun, Guifan

2013-01-01

203

Nox Family NADPH Oxidases in Mechano-Transduction: Mechanisms and Consequences  

PubMed Central

Abstract Significance: The majority of cells in a multi-cellular organism are continuously exposed to ever-changing physical forces. Mechano-transduction links these events to appropriate reactions of the cells involving stimulation of signaling cascades, reorganization of the cytoskeleton and alteration of gene expression. Recent Advances: Mechano-transduction alters the cellular redox balance and the formation of reactive oxygen species (ROS). Nicotine amide adenine dinucleotide reduced form (NADPH) oxidases of the Nox family are prominent ROS generators and thus, contribute to this stress-induced ROS formation. Critical Issues: Different types and patterns of mechano-stress lead to Nox-dependent ROS formation and Nox-mediated ROS formation contributes to cellular responses and adaptation to physical forces. Thereby, Nox enzymes can mediate vascular protection during physiological mechano-stress. Despite this, over-activation and induction of Nox enzymes and a subsequent substantial increase in ROS formation also promotes oxidative stress in pathological situations like disturbed blood flow or extensive stretch. Future Directions: Individual protein targets of Nox-mediated redox-signaling will be identified to better understand the specificity of Nox-dependent ROS signaling in mechano-transduction. Nox-inhibitors will be tested to reduce cellular activation in response to mechano-stimuli. Antioxid. Redox Signal. 20, 887–898. PMID:23682993

Weissmann, Norbert; Schroder, Katrin

2014-01-01

204

Thrombin Activates the Hypoxia-Inducible Factor1 Signaling Pathway in Vascular Smooth Muscle Cells : Role of the p22phox-Containing NADPH Oxidase  

Microsoft Academic Search

The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive

A. Gorlach; I. Diebold; V. B. Schini-Kerth; U. Berchner-Pfannschmidt; U. Roth; R. P. Brandes; T. Kietzmann; R. Busse

2001-01-01

205

NADPH oxidase 2-derived superoxide downregulates endothelial KCa3.1 in preeclampsia.  

PubMed

Endothelial dysfunction is associated with KCa3.1 dysfunction and contributes to the development of hypertension in preeclampsia. However, evidence of endothelial KCa3.1 dysfunction in the vascular system from women with preeclampsia is still lacking. Therefore, we examined whether endothelial KCa3.1 dysfunction occurs in vessels from women with preeclampsia. We compared KCa3.1 and NADPH oxidase (NOX) expression in umbilical vessels and primary cultured human umbilical vein endothelial cells (HUVECs) from normal (NP; n=17) and preeclamptic pregnancy (PE; n=19) and examined the effects of plasma from NP or PE on KCa3.1 and NOX2 expression in primary cultured HUVECs from NP or human uterine microvascular endothelial cells. The endothelial KCa3.1 was downregulated, and NOX2 was upregulated, in umbilical vessels and HUVECs from PE, compared with those from NP. In addition, HUVECs from PE showed a significant decrease in KCa3.1 current. Plasma from PE induced KCa3.1 down regulation, NOX2 upregulation, phosphorylated-p38 mitogen-activated protein kinase downregulation, and superoxide generation, and these effects were prevented by antioxidants (tempol or tiron), NOX2 inhibition, or anti-lectin-like oxidized low-density lipoprotein (LDL) receptor 1 (LOX1) antibody. Oxidized LDL and the superoxide donor xanthine/xanthine oxidase mixture induced KCa3.1 downregulation. In contrast, plasma from PE did not generate hydrogen peroxide, and the hydrogen peroxide donor tert-butylhydroperoxide induced KCa3.1 upregulation. These results provide the first evidence that plasma from PE generates superoxide via a LOX1-NOX2-mediated pathway and downregulates endothelial KCa3.1, which may contribute to endothelial dysfunction and vasculopathy in preeclampsia. This suggests KCa3.1as a novel target for patients with preeclampsia. PMID:23261940

Choi, Shinkyu; Kim, Ji Aee; Na, Hye-Young; Kim, Ji-Eun; Park, Seonghee; Han, Ki-Hwan; Kim, Young Ju; Suh, Suk Hyo

2013-04-01

206

The reactivity of ortho-methoxy-substituted catechol radicals with sulfhydryl groups: contribution for the comprehension of the mechanism of inhibition of NADPH oxidase by apocynin.  

PubMed

Redox processes are involved in the mechanism of action of NADPH oxidase inhibitors such as diphenyleneiodonium and apocynin. Here, we studied the structure-activity relationship for apocynin and analogous ortho-methoxy-substituted catechols as inhibitors of the NADPH oxidase in neutrophils and their reactivity with peroxidase. Aiming to alter the reduction potential, the ortho-methoxy-catechol moiety was kept constant and the substituents at para position related to the hydroxyl group were varied. Two series of compounds were employed: methoxy-catechols bearing electron-withdrawing groups (MC-W) such as apocynin, vanillin, 4-nitroguaiacol, 4-cyanoguaiacol, and methoxy-catechol bearing electron-donating groups (MC-D) such as 4-methylguaiacol and 4-ethylguaiacol. We found that MC-D were weaker inhibitors compared to MD-W. Furthermore, the radicals generated by oxidation of MC-W via MPO/H(2)O(2), but not for MC-D, were able to oxidize glutathione (GSH) as verified by the formation of thiyl radicals, depletion of GSH, and recycling of the ortho-methoxy-catechols during their oxidations. The capacity of oxidizing sulfhydryl (SH) groups was also verified when ovalbumin was incubated with MC-W, but not for MC-D. Since the effect of apocynin has been correlated with inactivation of the cytosolic fractions of the NADPH oxidase complex and its oxidation during the inhibitory process develops a special role in this process, we suggest that the close relationship between the reactivity of the radicals of MC-W compounds with thiol groups and their efficacy as NADPH oxidase inhibitor could be the chemical pathway behind the mechanism of action of apocynin and should be taken into account in the design of new and specific NADPH oxidase inhibitors. PMID:17544376

Kanegae, Marília P P; da Fonseca, Luiz Marcos; Brunetti, Iguatemy L; Silva, Sueli de Oliveira; Ximenes, Valdecir F

2007-08-01

207

The role of endothelial interleukin-8/NADPH oxidase 1 axis in sepsis  

PubMed Central

Sepsis is a generalized inflammatory disease, caused by the hyperinflammatory response of the host, rather than by invading organisms. Endothelial cells play a crucial role in the pathogenesis of sepsis. In this study, we investigated the effects of interleukin-8 (IL-8), a known neutrophil chemoattractant, on lipopolysaccharide (LPS) -induced reactive oxygen species (ROS) production by endothelial cells, and its significance in the pathogenesis of LPS-mediated sepsis. The results revealed that IL-8 directly induced ROS production in human umbilical vein endothelial cells (HUVECs), and also mediated LPS-induced ROS production by HUVECs. Stimulation of HUVECs by LPS strongly enhanced tissue factor expression, a hallmark of severe sepsis, which was suppressed by IL-8 knockdown. We further discovered that NADPH oxidase (Nox) 1 expression in LPS-stimulated HUVECs was markedly repressed by IL-8 knockdown, and Nox1 knockdown reduced tissue factor expression, suggesting that the LPS/IL-8 signalling in endothelial cells was predominantly mediated by Nox1. In conclusion, LPS stimulation of endothelial cells causes activation of the IL-8–Nox1 axis, enhances the production of ROS, and ultimately contributes to the progression of severe sepsis. PMID:20518825

Miyoshi, Takashi; Yamashita, Kouhei; Arai, Toshiyuki; Yamamoto, Kokichi; Mizugishi, Kiyomi; Uchiyama, Takashi

2010-01-01

208

Endothelin1-Mediated Increase in Reactive Oxygen Species and NADPH Oxidase Activity in Hearts of Aryl Hydrocarbon Receptor (AhR) Null Mice  

Microsoft Academic Search

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor characterized to play a role in detection and adaptation to environmental stimuli. Genetic deletion of the AhR results in cardiac hypertrophy that is mediated primarily by endothelin-1 (ET-1); ET-1 has been implicated in the elevation of reactive oxygen species (ROS) in the heart, which are thought to contribute to several

Amie K. Lund; Steven L. Peterson; Graham S. Timmins; Mary K. Walker

2005-01-01

209

Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane  

Microsoft Academic Search

Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation,\\u000a neutrophils produce superoxide anion ($${\\\\text{O}}_2 ^{ - .} $$), which generates other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical ($${\\\\text{OH}}^{ \\\\bullet } $$) and hypochlorous acid (HOCl), together with microbicidal peptides and proteases. The enzyme responsible for $${\\\\text{O}}_2

Jamel El-Benna; Pham My-Chan Dang; Marie-Anne Gougerot-Pocidalo

2008-01-01

210

Panaxydol induces apoptosis through an increased intracellular calcium level, activation of JNK and p38 MAPK and NADPH oxidase-dependent generation of reactive oxygen species  

Microsoft Academic Search

Panaxydol, a polyacetylenic compound derived from Panax ginseng roots, has been shown to inhibit the growth of cancer cells. In this study, we demonstrated that panaxydol induced apoptosis\\u000a preferentially in transformed cells with a minimal effect on non-transformed cells. Furthermore, panaxydol was shown to induce\\u000a apoptosis through an increase in intracellular Ca2+ concentration ([Ca2+]i), activation of JNK and p38 MAPK,

Joo Young Kim; Su-Jin Yu; Hyun Ju Oh; Ji Young Lee; Yongjin Kim; Jeongwon Sohn

2011-01-01

211

Activation of microglial NADPH oxidase is synergistic with glial iNOS expression in inducing neuronal death: a dual-key mechanism of inflammatory neurodegeneration  

E-print Network

identified based on morphology and at 16–18 DIV 76 ± 5% of the cells in the culture were CGC's. All experiments were undertaken in accordance with the UK Animals (Scientific Procedures) Act 1986. Activation of glia in neuronal-glial culturesPage 2 of 15 (page... , and mitochondria. Mol Neurobiol 2003, 27:325-355. 7. McGeer PL, McGeer EG: The inflammatory response system of brain: implications for therapy of Alzheimer and other neu- rodegenerative diseases. Brain Res Brain Res Rev 1995, 21:195-218. 8. Bal-Price A, Brown GC...

Mander, Palwinder K; Brown, Guy C

2005-09-12

212

Accessory cells with a veiled morphology and movement pattern generated from monocytes after avoidance of plastic adherence and of NADPH oxidase activation. A comparison with GM-CSF/IL-4-induced monocyte-derived dendritic cells.  

PubMed

Veiled cells (VC) present in afferent lymph transport antigen from the periphery to the draining lymph nodes. Although VC in lymph form a heterogeneous population, some of the cells clearly belong on morphological grounds to the Langerhans cell (LC)/ dendritic cell (DC) series. Here we show that culturing monocytes for 24 hrs while avoiding plastic adherence (polypropylene tubes) and avoiding the activation of NADPH oxidase (blocking agents) results in the generation of a population of veiled accessory cells. The generated VC were actively moving cells like lymph-borne VC in vivo. The monocyte (mo)-derived VC population existed of CD14(dim/-) and CD14(brighT) cells. Of these the CD14(dim/-) VC were as good in stimulating allogeneic T cell proliferation as immature DC (iDC) obtained after one week of adherent culture of monocytes in granulocyte-macrophage-colony stimulating factor (GM-CSF)/interleukin (IL)-4. This underscores the accessory cell function of the mo-derived CD14(dim/-) VC. Although the CD14(dim/-)VC had a modest expression of the DC-specific marker CD83 and were positive for S100, expression of the DC-specific markers CD1a, Langerin, DC-SIGN, and DC-LAMP were absent. This indicates that the here generated CD14(dim/-) VC can not be considered as classical LC/DC. It was also impossible to turn the CD14(dim/-) mo-derived VC population into typical DC by culture for one week in GM-CSF/IL-4 or LPS. In fact the cells died tinder such circumstances, gaining some macrophage characteristics before dying. The IL-12 production from mo-derived CD14(dim/-) VC was lower, whereas the production of IL-10 was higher as compared to iDC. Consequently the T cells that were stimulated by these mo-derived VC produced less IFN-gamma as compared with T cells stimulated by iDC. Our data indicate that it is possible to rapidly generate a population of CD14(dim/-) veiled accessory cells from monocytes. The marker pattern and cytokine production of these VC indicate that this population is not a classical DC population. The cells might earlier be related to the veiled macrophage-like cells also earlier described in afferent lymph. PMID:12182452

Ruwhof, Cindy; Canning, Martha O; Grotenhuis, Kristel; de Wit, Harm J; Florencia, Zenovia Z; de Haan-Meulman, Meeny; Drexhage, Hemmo A

2002-07-01

213

Evolutionary Dynamics of the Human NADPH Oxidase Genes CYBB, CYBA, NCF2, and NCF4: Functional Implications  

PubMed Central

The phagocyte NADPH oxidase catalyzes the reduction of O2 to reactive oxygen species with microbicidal activity. It is composed of two membrane-spanning subunits, gp91-phox and p22-phox (encoded by CYBB and CYBA, respectively), and three cytoplasmic subunits, p40-phox, p47-phox, and p67-phox (encoded by NCF4, NCF1, and NCF2, respectively). Mutations in any of these genes can result in chronic granulomatous disease, a primary immunodeficiency characterized by recurrent infections. Using evolutionary mapping, we determined that episodes of adaptive natural selection have shaped the extracellular portion of gp91-phox during the evolution of mammals, which suggests that this region may have a function in host-pathogen interactions. On the basis of a resequencing analysis of approximately 35 kb of CYBB, CYBA, NCF2, and NCF4 in 102 ethnically diverse individuals (24 of African ancestry, 31 of European ancestry, 24 of Asian/Oceanians, and 23 US Hispanics), we show that the pattern of CYBA diversity is compatible with balancing natural selection, perhaps mediated by catalase-positive pathogens. NCF2 in Asian populations shows a pattern of diversity characterized by a differentiated haplotype structure. Our study provides insight into the role of pathogen-driven natural selection in an innate immune pathway and sheds light on the role of CYBA in endothelial, nonphagocytic NADPH oxidases, which are relevant in the pathogenesis of cardiovascular and other complex diseases. PMID:23821607

Tarazona-Santos, Eduardo; Magalhaes, Wagner C.S.; Chen, Renee; Lyon, Fernanda; Burdett, Laurie; Crenshaw, Andrew; Fabbri, Cristina; Pereira, Latife; Pinto, Laelia; Redondo, Rodrigo A.F.; Sestanovich, Ben; Yeager, Meredith; Chanock, Stephen J.

2013-01-01

214

Androgens induce oxidative stress and radiation resistance in prostate cancer cells though NADPH oxidase  

PubMed Central

Androgen deprivation therapy (ADT) facilitates the response of prostate cancer (PC) to radiation. Androgens have been shown to induce elevated basal levels of reactive oxygen species (ROS) in PC, leading to adaptation to radiation-induced cytotoxic oxidative stress. Here, we show that androgens increase the expression of p22phox and gp91phox subunits of NADPH oxidase (NOX) and ROS production by NOX2 and NOX4 in PC. Pre-radiation treatment of 22Rv1 human PC cells with NOX inhibitors sensitize the cells to radiation similarly to ADT, suggesting that their future usage may spare the need for adjuvant ADT in PC patients undergoing radiation. PMID:19546883

Lu, J P; Monardo, L; Bryskin, I; Hou, Z F; Trachtenberg, J; Wilson, B C; Pinthus, J H

2009-01-01

215

An inhibitor of NADPH oxidase-4 attenuates established pulmonary fibrosis in a rodent disease model.  

PubMed

Idiopathic pulmonary fibrosis is a chronic progressive disease of increasing prevalence for which there is no effective therapy. Increased oxidative stress associated with an oxidant-antioxidant imbalance is thought to contribute to disease progression. NADPH oxidases (Nox) are a primary source of reactive oxygen species within the lung and cardiovascular system. We demonstrate that the Nox4 isoform is up-regulated in the lungs of patients with IPF and in a rodent model of bleomycin-induced pulmonary fibrosis and vascular remodeling. Nox4 is constitutively active, and therefore increased expression levels are likely to contribute to disease pathology. Using a small molecule Nox4/Nox1 inhibitor, we demonstrate that targeting Nox4 results in attenuation of an established fibrotic response, with reductions in gene transcripts for the extracellular matrix components collagen 1?1, collagen 3?1, and fibronectin and in principle pathway components associated with pulmonary fibrosis and hypoxia-mediated vascular remodeling: transforming growth factor (TGF)-?1, plasminogen activator inhibitor-1, hypoxia-inducible factor, and Nox4. TGF-?1 is a principle fibrotic mediator responsible for inducing up-regulation of profibrotic pathways associated with disease pathology. Using normal human lung-derived primary fibroblasts, we demonstrate that inhibition of Nox4 activity using a small molecule antagonist attenuates TGF-?1-mediated up-regulation in expression of profibrotic genes and inhibits the differentiation of fibroblast to myofibroblasts, that is associated with up-regulation in smooth muscle actin and acquisition of a contractile phenotype. These studies support the view that targeting Nox4 may provide a therapeutic approach for attenuating pulmonary fibrosis. PMID:23977848

Jarman, Elizabeth R; Khambata, Valerie S; Cope, Claire; Jones, Peter; Roger, Jan; Ye, Li Yun; Duggan, Nicholas; Head, Denise; Pearce, Andrew; Press, Neil J; Bellenie, Ben; Sohal, Bindi; Jarai, Gabor

2014-01-01

216

Inhibition of NADPH oxidase prevents acute lung injury in obese rats following severe trauma.  

PubMed

Lung capillary filtration coefficient (Kf) and impacts of oxidative stress have not been determined in the setting of severe trauma, especially in obese patients who exhibit increased lung injury. We hypothesized that severe trauma leads to a greater increase in lung Kf in obesity due to exacerbated production of and/or vulnerability to oxidative stress. Severe trauma was induced in lean and obese Zucker rats by muscle injury, fibula fracture, and bone component injection to both hindlimbs, with or without 24-h treatments of apocynin, a NADPH oxidase (NOX) inhibitor. Lung wet/dry weight ratios, lung vascular Kf, lung neutrophil counts, lung NOX and myeloperoxidase (MPO) activity, and plasma IL-6 levels were measured 24 h after trauma. In an additional study, lungs were isolated from nontrauma lean and obese rats to determine the acute effect of phenazime methosulfate, a superoxide donor, on pulmonary vascular Kf. After trauma, compared with lean rats, obese rats exhibited greater increases in lung capillary Kf, neutrophil accumulation, NOX and MPO activity, and plasma IL-6. The lung wet/dry weight ratio was increased in obese rats but not in lean rats. Apocynin treatment decreased lung Kf, neutrophil counts, NOX and MPO activities, wet/dry weight ratio, and plasma IL-6 in obese rats. Phenazime methosulfate treatment resulted in a greater increase in lung Kf in nontrauma obese rats compared with nontrauma lean rats. These results suggest that obese rats are susceptible to lung injury following severe trauma due to increased production of and responsiveness to pulmonary oxidative stress. PMID:24414071

Xiang, Lusha; Lu, Silu; Mittwede, Peter N; Clemmer, John S; Hester, Robert L

2014-03-01

217

Molecular evolution of Phox-related regulatory subunits for NADPH oxidase enzymes  

PubMed Central

Background The reactive oxygen-generating NADPH oxidases (Noxes) function in a variety of biological roles, and can be broadly classified into those that are regulated by subunit interactions and those that are regulated by calcium. The prototypical subunit-regulated Nox, Nox2, is the membrane-associated catalytic subunit of the phagocyte NADPH-oxidase. Nox2 forms a heterodimer with the integral membrane protein, p22phox, and this heterodimer binds to the regulatory subunits p47phox, p67phox, p40phox and the small GTPase Rac, triggering superoxide generation. Nox-organizer protein 1 (NOXO1) and Nox-activator 1 (NOXA1), respective homologs of p47phox and p67phox, together with p22phox and Rac, activate Nox1, a non-phagocytic homolog of Nox2. NOXO1 and p22phox also regulate Nox3, whereas Nox4 requires only p22phox. In this study, we have assembled and analyzed amino acid sequences of Nox regulatory subunit orthologs from vertebrates, a urochordate, an echinoderm, a mollusc, a cnidarian, a choanoflagellate, fungi and a slime mold amoeba to investigate the evolutionary history of these subunits. Results Ancestral p47phox, p67phox, and p22phox genes are broadly seen in the metazoa, except for the ecdysozoans. The choanoflagellate Monosiga brevicollis, the unicellular organism that is the closest relatives of multicellular animals, encodes early prototypes of p22phox, p47phox as well as the earliest known Nox2-like ancestor of the Nox1-3 subfamily. p67phox- and p47phox-like genes are seen in the sea urchin Strongylocentrotus purpuratus and the limpet Lottia gigantea that also possess Nox2-like co-orthologs of vertebrate Nox1-3. Duplication of primordial p47phox and p67phox genes occurred in vertebrates, with the duplicated branches evolving into NOXO1 and NOXA1. Analysis of characteristic domains of regulatory subunits suggests a novel view of the evolution of Nox: in fish, p40phox participated in regulating both Nox1 and Nox2, but after the appearance of mammals, Nox1 (but not Nox2) became independent of p40phox. In the fish Oryzias latipes, a NOXO1 ortholog retains an autoinhibitory region that is characteristic of mammalian p47phox, and this was subsequently lost from NOXO1 in later vertebrates. Detailed amino acid sequence comparisons identified both putative key residues conserved in characteristic domains and previously unidentified conserved regions. Also, candidate organizer/activator proteins in fungi and amoeba are identified and hypothetical activation models are suggested. Conclusion This is the first report to provide the comprehensive view of the molecular evolution of regulatory subunits for Nox enzymes. This approach provides clues for understanding the evolution of biochemical and physiological functions for regulatory-subunit-dependent Nox enzymes. PMID:17900370

Kawahara, Tsukasa; Lambeth, J David

2007-01-01

218

Ebselen and congeners inhibit NADPH-oxidase 2 (Nox2)-dependent superoxide generation by interrupting the binding of regulatory subunits  

PubMed Central

Summary NADPH-oxidases are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits. PMID:22726689

Smith, Susan M.E.; Min, Jaeki; Ganesh, Thota; Diebold, Becky; Kawahara, Tsukasa; Zhu, Yerun; McCoy, James; Sun, Aiming; Snyder, James P.; Fu, Haian; Du, Yuhong; Lewis, Iestyn; Lambeth, J. David

2012-01-01

219

Phagocyte-like NADPH oxidase [Nox2] in cellular dysfunction in models of glucolipotoxicity and diabetes.  

PubMed

Increased intracellular generation of reactive oxygen species [ROS] has been implicated in the pathology of metabolic [diabetes] and neurodegenerative [Alzheimer's] diseases. Accumulating evidence suggests NADPH oxidases [Noxs] as the principal source for cellular ROS in humans. Of this class of enzymes, the phagocyte-like Nox [Nox2] has come under intense scrutiny as one of the "culprits" for the induction of cellular damage culminating in the onset of diabetes and its complications. Functional regulation of Nox2 is fairly complex due to its membranous [gp91(phox), p22(phox)] and cytosolic [p40(phox), p47(phox), p67(phox) and Rac1] cores, which require specific post-translational modification steps [phosphorylation and lipidation] for their membrane association. Therefore, optimal efficacy of Nox2 depends upon precise regulation of these signaling steps followed by translocation of the cytosolic components to the membrane. Interestingly, numerous recent studies have reported sustained activation of Nox2, ROS-derived oxidative stress, and cellular dysfunction in in vitro and in vivo models of glucolipotoxicity and diabetes. These investigations employed a variety of cell-permeable peptides and pharmacological inhibitors to impede Nox2 holoenzyme assembly and activation in pancreatic islet ?-cells, cardiomyocytes and retinal endothelial cells under conditions of glucolipotoxicity and diabetes. Herein, we highlight the existing evidence to implicate Nox2 as the "trigger" of cellular damage, and identify critical gaps in our current understanding that need to be addressed to further affirm the roles of Nox2 as a potential therapeutic target for the treatment of diabetes and other metabolic disorders. PMID:24462914

Kowluru, Anjaneyulu; Kowluru, Renu A

2014-04-01

220

NADPH Oxidase 1-Mediated Oxidative Stress Leads to Dopamine Neuron Death in Parkinson's Disease  

PubMed Central

Abstract Aim: Oxidative stress has long been considered as a major contributing factor in the pathogenesis of Parkinson's disease. However, molecular sources for reactive oxygen species in Parkinson's disease have not been clearly elucidated. Herein, we sought to investigate whether a superoxide-producing NADPH oxidases (NOXs) are implicated in oxidative stress-mediated dopaminergic neuronal degeneration. Results: Expression of various Nox isoforms and cytoplasmic components were investigated in N27, rat dopaminergic cells. While most of Nox isoforms were constitutively expressed, Nox1 expression was significantly increased after treatment with 6-hydroxydopamine. Rac1, a key regulator in the Nox1 system, was also activated. Striatal injection of 6-hydroxydopamine increased Nox1 expression in dopaminergic neurons in the rat substantia nigra. Interestingly, it was localized into the nucleus, and immunostaining for DNA oxidative stress marker, 8-oxo-dG, was increased. Nox1expression was also found in the nucleus of dopaminergic neurons in the substantia nigra of Parkinson's disease patients. Adeno-associated virus-mediated Nox1 knockdown or Rac1 inhibition reduced 6-hydroxydopamine-induced oxidative DNA damage and dopaminergic neuronal degeneration significantly. Innovation: Nox1/Rac1 could serve as a potential therapeutic target for Parkinson's disease. Conclusion: We provide evidence that dopaminergic neurons are equipped with the Nox1/Rac1 superoxide-generating system. Stress-induced Nox1/Rac1 activation causes oxidative DNA damage and neurodegeneration. Reduced dopaminergic neuronal death achieved by targeting Nox1/Rac1, emphasizes the impact of oxidative stress caused by this system on the pathogenesis and therapy in Parkinson's disease. Antioxid. Redox Signal. 16, 1033–1045. PMID:22098189

Choi, Dong-Hee; Cristovao, Ana Clara; Guhathakurta, Subhrangshu; Lee, Jongmin; Joh, Tong H.; Beal, M. Flint

2012-01-01

221

Assembly of the phagocyte NADPH oxidase: binding of Src homology 3 domains to proline-rich targets.  

PubMed Central

The NADPH oxidase responsible for generation of superoxide anion and related microbicidal oxidants by phagocytes is assembled from at least five distinct proteins. Two are cytosolic components (p47-phox and p67-phox) that contain Src homology 3 (SH3) domains and associate with a transmembrane cytochrome b558 upon activation. We show here that the SH3 domains of p47-phox bind to proline-rich sequences in p47-phox itself and the p22-phox subunit of cytochrome b558. Binding of the p47-phox SH3 domains to p22-phox was abolished by a mutation in one proline-rich sequence (Pro156-->Gln) noted in a distinct form of chronic granulomatous disease and was inhibited by a short proline-rich synthetic peptide corresponding to residues 149-162 of p22-phox. Expression of mutated p22-phox did not restore oxidase activity to p22-phox-deficient B cells and did not enable p22-phox-dependent translocation of p47-phox to membranes in phorbol ester-stimulated cells. We also show that the cytosolic oxidase components associate with one another through the C-terminal SH3 domain of p67-phox and a proline-rich C-terminal sequence in p47-phox. These SH3 target sites conform to consensus features deduced from SH3 binding sites in other systems. We propose a model in which the oxidase complex assembles through a mechanism involving SH3 domains of both cytosolic proteins and cognate proline-rich targets in other oxidase components. Images PMID:7938008

Leto, T L; Adams, A G; de Mendez, I

1994-01-01

222

Glutathione attenuates ethanol-induced alveolar macrophage oxidative stress and dysfunction by downregulating NADPH oxidases.  

PubMed

Chronic alcohol abuse increases lung oxidative stress and susceptibility to respiratory infections by impairing alveolar macrophage (AM) function. NADPH oxidases (Nox) are major sources of reactive oxygen species in AMs. We hypothesized that treatment with the critical antioxidant glutathione (GSH) attenuates chronic alcohol-induced oxidative stress by downregulating Noxes and restores AM phagocytic function. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from male C57BL/6J mice (8-10 wk) treated ± ethanol in drinking water (20% wt/vol, 12 wk) ± orally gavaged GSH in methylcellulose vehicle (300 mg x kg(-1) x day(-1), during week 12). MH-S cells, a mouse AM cell line, were treated ± ethanol (0.08%, 3 days) ± GSH (500 ?M, 3 days or last 1 day of ethanol). BAL and AMs were also isolated from ethanol-fed and control mice ± inoculated airway Klebsiella pneumoniae (200 colony-forming units, 28 h) ± orally gavaged GSH (300 mg/kg, 24 h). GSH levels (HPLC), Nox mRNA (quantitative RT-PCR) and protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol decreased GSH levels, increased Nox expression and activity, enhanced oxidative stress, impaired phagocytic function in AMs in vivo and in vitro, and exacerbated K. pneumonia-induced oxidative stress. Although how oral GSH restored GSH pools in ethanol-fed mice is unknown, oral GSH treatments abrogated the detrimental effects of chronic alcohol exposure and improved AM function. These studies provide GSH as a novel therapeutic approach for attenuating alcohol-induced derangements in AM Nox expression, oxidative stress, dysfunction, and risk for pneumonia. PMID:24441868

Yeligar, Samantha M; Harris, Frank L; Hart, C Michael; Brown, Lou Ann S

2014-03-01

223

NADPH Oxidase 1, a Novel Molecular Source of ROS in Hippocampal Neuronal Death in Vascular Dementia  

PubMed Central

Abstract Aims: Chronic cerebral hypoperfusion (CCH) is a common pathological factor that contributes to neurodegenerative diseases such as vascular dementia (VaD). Although oxidative stress has been strongly implicated in the pathogenesis of VaD, the molecular mechanism underlying the selective vulnerability of hippocampal neurons to oxidative damage remains unknown. We assessed whether the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, a specialized superoxide generation system, plays a role in VaD by permanent ligation of bilateral common carotid arteries in rats. Results: Male Wistar rats (10 weeks of age) were subjected to bilateral occlusion of the common carotid arteries (two-vessel occlusion [2VO]). Nox1 expression gradually increased in hippocampal neurons, starting at 1 week after 2VO and for approximately 15 weeks after 2VO. The levels of superoxide, DNA oxidation, and neuronal death in the CA1 subfield of the hippocampus, as well as consequential cognitive impairment, were increased in 2VO rats. Both inhibition of Nox by apocynin, a putative Nox inhibitor, and adeno-associated virus-mediated Nox1 knockdown significantly reduced 2VO-induced reactive oxygen species generation, oxidative DNA damage, hippocampal neuronal degeneration, and cognitive impairment. Innovation and Conclusion: We provided evidence that neuronal Nox1 is activated in the hippocampus under CCH, causing oxidative stress and consequential hippocampal neuronal death and cognitive impairment. This evidence implies that Nox1-mediated oxidative stress plays an important role in neuronal cell death and cognitive dysfunction in VaD. Nox1 may serve as a potential therapeutic target for VaD. Antioxid. Redox Signal. 21, 533–550. PMID:24294978

Choi, Dong-Hee; Lee, Kyoung-Hee; Kim, Ji-Hye; Seo, Ju-Ha; Kim, Hahn Young; Shin, Chan Young; Han, Jung-Soo; Han, Seol-Heui; Kim, Yoon-Seong

2014-01-01

224

Role of NADPH Oxidase in Formation and Function of Multinucleated Giant Cells  

PubMed Central

Macrophages play essential roles in a wide variety of physiological and pathological processes. One of the unique features of these phagocytic leukocytes is their ability to fuse, forming multinucleated giant cells. Multinucleated giant cells are important mediators of tissue remodeling and repair and are also responsible for removal or sequestration of foreign material, intracellular bacteria and non-phagocytosable pathogens, such as parasites and fungi. Depending on the tissue where fusion occurs and the inflammatory insult, multinucleated giant cells assume distinctly different phenotypes. Nevertheless, the ultimate outcome is the formation of large cells that can resorb bone tissue (osteoclasts) or foreign material and pathogens (giant cells) extracellularly. While progress has been made in recent years, the mechanisms and factors involved in macrophage fusion are still not fully understood. In addition to cytokines and a number of adhesion proteins and receptors, it is becoming increasingly clear that NADPH oxidase-generated reactive oxygen species (ROS) also play an important role in macrophage fusion. In this review, we provide an overview of macrophage multinucleation, with a specific focus on the role of NADPH oxidases and ROS in macrophage fusion and in the function of multinucleated giant cells. In addition, we provide an updated overview of the role of these cells in inflammation and various autoimmune diseases. PMID:20375608

Quinn, Mark T.; Schepetkin, Igor A.

2009-01-01

225

Nitroglycerine-induced nitrate tolerance compromises propofol protection of the endothelial cells against TNF-?: the role of PKC-?2 and NADPH oxidase.  

PubMed

Continuous treatment with organic nitrates causes nitrate tolerance and endothelial dysfunction, which is involved with protein kinase C (PKC) signal pathway and NADPH oxidase activation. We determined whether chronic administration with nitroglycerine compromises the protective effects of propofol against tumor necrosis factor (TNF-) induced toxicity in endothelial cells by PKC- ?2 dependent NADPH oxidase activation. Primary cultured human umbilical vein endothelial cells were either treated or untreated with TNF- ? (40 ng/mL) alone or in the presence of the specific PKC- ?2 inhibitor CGP53353 (1 ?M)), nitroglycerine (10 ?M), propofol (100 ?M), propofol plus nitroglycerin, or CGP53353 plus nitroglycerine, respectively, for 24 hours. TNF-? increased the levels of superoxide, Nox (nitrate and nitrite), malondialdehyde, and nitrotyrosine production, accompanied by increased protein expression of p-PKC-?2, gP91phox, and endothelial cell apoptosis, whereas all these changes were further enhanced by nitroglycerine. CGP53353 and propofol, respectively, reduced TNF-? induced oxidative stress and cell toxicity. CGP53353 completely prevented TNF- ? induced oxidative stress and cell toxicity in the presence or absence of nitroglycerine, while the protective effects of propofol were neutralized by nitroglycerine. It is concluded that nitroglycerine comprises the protective effects of propofol against TNF-? stimulation in endothelial cells, primarily through PKC-?2 dependent NADPH oxidase activation. PMID:24396568

Lei, Shaoqing; Su, Wating; Liu, Huimin; Xu, Jinjin; Xia, Zhong-yuan; Yang, Qing-jun; Qiao, Xin; Du, Yun; Zhang, Liangqing; Xia, Zhengyuan

2013-01-01

226

Mild exposure of RIN-5F ?-cells to human islet amyloid polypeptide aggregates upregulates antioxidant enzymes via NADPH oxidase-RAGE: An hormetic stimulus?  

PubMed Central

The presence of amyloid aggregates of human islet amyloid polypeptide (hIAPP), a hallmark of type 2 diabetes, contributes to pancreatic ?-cell impairment, where oxidative stress plays a key role. A contribution of NADPH oxidase to reactive oxygen species (ROS) generation after cell exposure to micromolar concentrations of hIAPP aggregates has been suggested. However, little is known about ?-cells exposure to lower amounts of hIAPP aggregates, similar to those found in human pancreas. Thus, we aimed to investigate the events resulting from RIN-5F cells exposure to nanomolar concentrations of toxic hIAPP aggregates. We found an early and transient rise of NADPH oxidase activity resulting from increased Nox1 expression following the engagement of receptor for advanced glycation end-products (RAGE) by hIAPP aggregates. Unexpectedly, NADPH oxidase activation was not accompanied by a significant ROS increase and the lipoperoxidation level was significantly reduced. Indeed, cell exposure to hIAPP aggregates affected the antioxidant defences, inducing a significant increase of the expression and activity of catalase and glutathione peroxidase. We conclude that exposure of pancreatic ?-cells to nanomolar concentrations of hIAPP aggregates for a short time induces an hormetic response via the RAGE-Nox1 axis; the latter stimulates the enzymatic antioxidant defences that preserve the cells against oxidative stress damage. PMID:24416718

Borchi, Elisabetta; Bargelli, Valentina; Guidotti, Valentina; Berti, Andrea; Stefani, Massimo; Nediani, Chiara; Rigacci, Stefania

2013-01-01

227

Atorvastatin attenuates homocysteine-induced apoptosis in human umbilical vein endothelial cells via inhibiting NADPH oxidase-related oxidative stress-triggered p38MAPK signaling  

PubMed Central

Aim: To examine the effect of atorvastatin on homocysteine (Hcy)-induced reactive oxygen species (ROS) production and apoptosis in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were cultured with Hcy (0.1?5 mmol/L) in the presence or absence of atorvastatin (1?100 ?mol//L) or various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI, 10 ?mol/L), the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 (10 ?mol/L) and antioxidants N-acetyl cysteine (NAC, 1 mmol/L). Cell apoptosis was evaluated by Annexin V/propidium iodide staining and flow cytometry. ROS were detected by 2?,7?-dichlorodihydrofluorescein diacetate (H2DCFH-DA). NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. Hcy-induced expression of p38MAPK protein was measured by Western blotting analysis. Results: Atorvastatin inhibited endothelial cell apoptosis induced by 1 mmol/L Hcy in a dose-dependent manner and the maximal inhibitory effect was reached at 100 ?mol/L. Atorvastatin (10 ?mol/L) significantly suppressed Hcy (1 mmol/L for 30 min) induced ROS accumulation (3.17±0.33 vs 4.34±0.31, P<0.05). Atorvastatin (10 ?mol/L) also antagonized Hcy (1 mmol/L for 30 min) induced activation of NADPH oxidase (2.57±0.49 vs 3.33±0.6, P<0.05). Furthermore, atorvastatin inhibited Hcy-induced phosphorylation of p38 MAPK (1.7±0.1 vs 2.22±0.25, P<0.05), similar effects occurred with DPI, NAC and SB203580. Conclusion: Atorvastatin may inhibit Hcy-induced ROS accumulation and endothelium cell apoptosis through an NADPH oxidase and/or p38MAPK-dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effect on endothelial function. PMID:19767766

Bao, Xiao-mei; Wu, Chun-fang; Lu, Guo-ping

2009-01-01

228

Pigment Epithelium-Derived Factor Inhibits Neointimal Hyperplasia after Vascular Injury by Blocking NADPH Oxidase-Mediated Reactive Oxygen Species Generation  

PubMed Central

Pigment epithelium-derived factor (PEDF) inhibits cytokine-induced endothelial cell activation through its antioxidative properties. However, the effect of PEDF on restenosis remains to be elucidated. Because the pathophysiological feature of restenosis is characterized by increased superoxide formation and accumulation of smooth muscle cells (SMCs), PEDF may inhibit this process via suppression of reactive oxygen species generation. We investigated here whether PEDF could prevent neointimal formation after balloon injury. PEDF levels were decreased in balloon-injured arteries. Adenoviral vector encoding human PEDF (Ad-PEDF) prevented neointimal formation. Expression and superoxide generation of the membrane components of NADPH oxidase, p22phox and gp91phox, in the neointima were also suppressed by Ad-PEDF. Ad-PEDF reduced G1 cyclin (cyclin D1 and E) expression and increased p27, a cyclin-dependent kinase inhibitor. In vitro, PEDF inhibited platelet-derived growth factor-BB-induced SMC proliferation and migration by blocking reactive oxygen species generation through suppression of NADPH oxidase activity via down-regulation of p22PHOX and gp91PHOX. PEDF down-regulated G1 cyclins and up-regulated p27 levels in platelet-derived growth factor-BB-exposed SMCs as well. These results demonstrate that PEDF could inhibit neointimal formation via suppression of NADPH oxidase-mediated reactive oxygen species generation. Our present study suggests that substitution of PEDF may be a novel therapeutic strategy for restenosis after balloon angioplasty. PMID:17525281

Nakamura, Kazuo; Yamagishi, Sho-ichi; Matsui, Takanori; Yoshida, Takafumi; Takenaka, Katsuhiko; Jinnouchi, Yuko; Yoshida, Yumiko; Ueda, Shin-ichiro; Adachi, Hisashi; Imaizumi, Tsutomu

2007-01-01

229

Angiotensin II-dependent chronic hypertension and cardiac hypertrophy are unaffected by gp91phox-containing NADPH oxidase.  

PubMed

The gp91phox-containing NADPH oxidase is the major source of reactive oxygen species (ROS) in the cardiovascular system and inactivation of gp91phox has been reported to blunt hypertension and cardiac hypertrophy seen in angiotensin (Ang) II-infused animals. In the current study, we sought to determine the role of gp91phox-derived ROS on cardiovascular outcomes of chronic exposure to Ang II. The gp91phox-deficient mice were crossed with transgenic mice expressing active human renin in the liver (TTRhRen). TTRhRen mice exhibit chronic Ang II-dependent hypertension and frank cardiac hypertrophy by age 10 to 12 weeks. Four genotypes of mice were generated: control, TTRhRen trangenics (TTRhRen), gp91phox-deficient (gp91-), and TTRhRen transgenic gp91phox-deficient (TTRhRen/gp91-). Eight to 10 mice/group were studied. ROS levels were significantly reduced (P<0.05) in the heart and aorta of TTRhRen/gp91- and gp91-mice compared with control counterparts, and this was associated with reduced cardiac, aortic, and renal NADPH oxidase activity (P<0.05). Systolic blood pressure (SBP), cardiac mass, and cardiac fibrosis were increased in TTRhRen versus controls. In contrast to its action on ROS generation, gp91phox inactivation had no effect on development of hypertension or cardiac hypertrophy in TTRhRen mice, although interstitial fibrosis was reduced. Cardiac and renal expression of gp91phox homologues, Nox1 and Nox4, was not different between groups. Thus, although eliminating gp91phox-associated ROS production may be important in cardiovascular consequences in acute insult models, it does not prevent the development of hypertension and cardiac hypertrophy in a model in which the endogenous renin-angiotensin system is chronically upregulated. PMID:15753233

Touyz, Rhian M; Mercure, Chantel; He, Ying; Javeshghani, Danesh; Yao, Guoying; Callera, Glaucia E; Yogi, Alvaro; Lochard, Nadheige; Reudelhuber, Timothy L

2005-04-01

230

Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans  

Microsoft Academic Search

Summary NADPH oxidases (Nox) have been characterized as higher eukaryotic enzymes used deliberately to pro- duce reactive oxygen species (ROS). The recent dis- covery of new functional members of the Nox family in plants and animals has led to the recognition of the increasing importance of ROS as signals involved in regulation of diverse cellular processes such as defence, growth

Teresa Lara-Ortíz; Héctor Riveros-Rosas; Jesús Aguirre

231

Genetic Deficiency of NADPH Oxidase Does Not Diminish but Rather Enhances LPS-Induced Acute Inflammatory Responses in Vivo  

PubMed Central

Reactive oxygen species (ROS) and oxidative stress are thought to play a central role in the etiology of cell dysfunction and tissue damage in sepsis. However, there is limited and controversial evidence from in vivo studies that ROS mediate cell signaling processes that elicit acute inflammatory responses during sepsis. Since NADPH oxidase is one of the main cellular sources of ROS, we investigated the role of this enzyme in lipopolysaccharide (LPS)-induced acute inflammation in vivo, utilizing mice deficient in the gp91phox or p47phox subunits of NADPH oxidase. Age and body-weight matched C57BL/6J wild-type (WT) and gp91phox?/? and p47phox?/? mice were injected i.p. with 50 µg LPS or saline vehicle, and sacrificed at different time points up to 24 hours. We found that LPS-induced acute inflammatory responses in serum and tissues were not significantly diminished in gp91phox?/? and p47phox?/? mice compared to WT mice. Rather, genetic deficiency of NADPH oxidase was associated with enhanced gene expression of inflammatory mediators and increased neutrophil recruitment to lung and heart. Furthermore, no protection from LPS-induced septic death was observed in either knockout strain. Our findings suggest that NADPH oxidase-mediated ROS production and cellular redox signaling do not promote but instead limit LPS-induced acute inflammatory responses in vivo. PMID:19124074

Zhang, Wei-Jian; Wei, Hao; Frei, Balz

2008-01-01

232

Involvement of an NAD(P)H oxidase-like enzyme in superoxide anion and hydrogen peroxide generation by rat type II cells  

PubMed Central

BACKGROUND: Although alveolar macrophages are considered to be the primary cellular mediators of host defence in the lung, there is increasing evidence that type II cells may also play an active role in host defence. A study was undertaken to investigate whether type II cells generate O2-. and H2O2 via an NADPH oxidase-like system and whether exposure of the type II cells to soluble or particulate stimuli known to activate NADPH oxidase in macrophages also leads to increased production of H2O2. METHODS: Rat type II cells and alveolar macrophages were exposed to 10, 100, or 1000 nM phorbol-12-myristate-13-acetate (PMA) and the production of O2-. and H2O2 was determined by chemiluminescence. Thirty minutes before stimulation with 1 microM PMA type II cells were also exposed to the same concentrations of a protein kinase C (PKC) antagonist GF109203x, the non-selective protein kinase inhibitor staurosporine (1, 10, or 100 nM), or the NADPH oxidase inhibitor diphenyliodonium chloride (DPI) (1, 10, 100, or 1000 microM). The effects of arachidonic acid, zymosan and Staphylococcus aureus on H2O2 production were determined. Cell membrane fractions from type II cells and macrophages were assayed for NADPH oxidase activity. RESULTS: After exposure to 1 microM PMA, O2-. and H2O2 generation increased 6.3- fold and 9.0-fold, respectively, in type II cells and 2.4-fold and 5.2- fold, respectively, in macrophages. In contrast to the macrophages, the increase in O2-. and H2O2 generation by type II cells was completely prevented by 1 mM KCN. Preexposure to GF109203x, staurosporine, or DPI completely prevented the rise in O2-. and H2O2 generation. Mean (SD) NADPH oxidase activity of 138 (38) nmol O2-./min/mg protein was found in membrane fraction I of the type II cells, and 102 (31) nmol O2- ./min/mg protein in fraction II. Macrophages showed higher NADPH oxidase activity in membrane fraction II. In type II cells exposure to arachidonic acid led to a significant 5.3-fold increase in H2O2 generation, exposure to zymosan increased H2O2 generation 46-fold, and exposure to S aureus 25-fold with a maximum 30-50 minutes after addition of the bacteria. CONCLUSIONS: Type II cells generate O2-. and H2O2 via a PKC-mediated activation of an NAD(P)H oxidase-like membrane bound enzyme. Arachidonic acid, zymosan, and bacteria also give rise to increased H2O2 production. Type II cells might thus play an active role in host defence. ??? PMID:9176540

van Klaveren, R. J.; Roelant, C.; Boogaerts, M.; Demedts, M.; Nemery, B.

1997-01-01

233

In Silico Sequence Analysis Reveals New Characteristics of Fungal NADPH Oxidase Genes  

PubMed Central

NADPH oxidases (Noxes), transmembrane proteins found in most eukaryotic species, generate reactive oxygen species and are thereby involved in essential biological processes. However, the fact that genes encoding ferric reductases and ferric-chelate reductases share high sequence similarities and domains with Nox genes represents a challenge for bioinformatic approaches used to identify Nox-encoding genes. Further, most studies on fungal Nox genes have focused mainly on functionality, rather than sequence properties, and consequently clear differentiation among the various Nox isoforms has not been achieved. We conducted an extensive sequence analysis to identify putative Nox genes among 34 eukaryotes, including 28 fungal genomes and one Oomycota genome. Analyses were performed with respect to phylogeny, transmembrane helices, di-histidine distance and glycosylation. Our analyses indicate that the sequence properties of fungal Nox genes are different from those of human and plant Nox genes, thus providing novel insight that will enable more accurate identification and characterization of fungal Nox genes.

Detry, Nicolas; Choi, Jaeyoung; Kuo, Hsiao-Che; Asiegbu, Fred O.

2014-01-01

234

Catechol oxidase — structure and activity  

Microsoft Academic Search

Recently determined structures of copper-containing plant catechol oxidase in three different catalytic states have provided new insights into the mechanism of this enzyme and its relationship to other copper type-3 proteins. Moreover, the active site of catechol oxidase has been found to be structurally conserved with the oxygen-binding site of a molluscan hemocyanin.

Christoph Eicken; Bernt Krebs; James C Sacchettini

1999-01-01

235

Which NADPH Oxidase Isoform Is Relevant for Ischemic Stroke? The Case for Nox 2  

PubMed Central

Abstract Significance and Recent Advances: Ischemic stroke is the leading cause of disability and third in mortality in industrialized nations. Immediate restoration of cerebral blood flow is crucial to salvage brain tissue, but only few patients are eligible for recanalization therapy. Thus, the need for alternative neuroprotective strategies is huge, and antioxidant interventions have long been studied in this context. Reactive oxygen species (ROS) physiologically serve as signaling molecules, but excessive amounts of ROS, as generated during ischemia/reperfusion (I/R), contribute to tissue injury. Critical Issues: Nevertheless and despite a strong rational of ROS being a pharmacological target, all antioxidant interventions failed to improve functional outcome in human clinical trials. Antioxidants may interfere with physiological functions of ROS or do not reach the crucial target structures of ROS-induced injury effectively. Future Directions: Thus, a potentially more promising approach is the inhibition of the source of disease-promoting ROS. Within recent years, NADPH oxidases (Nox) of the Nox family have been identified as mediators of neuronal pathology. As, however, several Nox homologs are expressed in neuronal tissue, and as many of the pharmacological inhibitors employed are rather unspecific, the concept of Nox as mediators of brain damage is far from being settled. In this review, we will discuss the contribution of Nox homologs to I/R injury at large as well as to neuronal damage in particular. We will illustrate that the current data provide evidence for Nox2 as the most important NADPH oxidase mediating cerebral injury. Antioxid. Redox Signal. 18, 1400–1417. PMID:22746273

Kahles, Timo

2013-01-01

236

NADPH oxidase inhibitors: a decade of discovery from Nox2ds to HTS.  

PubMed

NADPH oxidases (Nox) are established as major sources of reactive oxygen species (ROS). Over the past two decades, Nox-derived ROS have emerged as pivotal in the development of myriad diseases involving oxidative stress. In contrast, Nox are also involved in signaling mechanisms necessary for normal cell function. The study of these enzymes in physiological and pathophysiological conditions is made considerably more complex by the discovery of 7 isoforms: Nox1 through 5 as well as Duox1 and 2, each with its own specific cytosolic components, regulatory control mechanisms, subcellular localization and/or tissue distribution. A clear understanding of the role individual isoforms play in a given system is hindered by the lack of isoform-specific inhibitors. In animal models, knockdown or knockout methodologies are providing definitive answers to perplexing questions of the complex interplay of multiple Nox isoforms in cell and tissue signaling. However, the complex structures and interactions of these heteromeric isozymes predict pleiotropic actions of the Nox subunits and thus suppression of these proteins is almost certain to have untoward effects. Thus, as both therapies and pharmacological tools, molecule-based inhibitors continue to prove extremely useful and rational in design. Unfortunately, many of the available inhibitors have proven non-specific, falling into the category of scavengers or inhibitors of more than one source of ROS. Here, we will review some of the efforts that have been undertaken to develop specific inhibitors of NADPH oxidase over the past decade, from the peptidic inhibitor Nox2ds-tat to more recent small molecule inhibitors that have emerged from high-throughput screening campaigns. PMID:22585059

Cifuentes-Pagano, Eugenia; Csanyi, Gabor; Pagano, Patrick J

2012-07-01

237

Similar and distinct roles of NADPH oxidase components in the tangerine pathotype of Alternaria alternata.  

PubMed

The fungal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, which has been implicated in the production of low-level reactive oxygen species (ROS), contains mainly NoxA, NoxB (gp91(phox) homologues) and NoxR (p67(phox) homologue). Here, we report the developmental and pathological functions of NoxB and NoxR in the tangerine pathotype of Alternaria alternata. Loss-of-function genetics revealed that all three Nox components are required for the accumulation of cellular hydrogen peroxide (H?O?). Alternaria alternata strains lacking NoxA, NoxB or NoxR also displayed an increased sensitivity to H?O? and many ROS-generating oxidants. These phenotypes are highly similar to those previously seen for the ?yap1 mutant lacking a YAP1 transcriptional regulator and for the ?hog1 mutant lacking a HOG1 mitogen-activated protein (MAP) kinase, implicating a possible link among them. A fungal strain carrying a NoxA NoxB or NoxA NoxR double mutation was more sensitive to the test compounds than the strain mutated at a single gene, implicating a synergistic function among Nox components. The ?noxB mutant strain failed to produce any conidia; both ?noxA and ?noxR mutant strains showed a severe reduction in sporulation. Mutant strains carrying defective NoxB had higher chitin content than the wild-type and were insensitive to calcofluor white, Congo red and the fungicides vinclozolin and fludioxonil. Virulence assays revealed that all three Nox components are required for the elaboration of the penetration process. The inability to penetrate the citrus host, observed for ?nox mutants, could be overcome by wounding and by reacquiring a dominant Nox gene. The A.?alternata?NoxR did not influence the expression of NoxB, but negatively regulated NoxA. Importantly, the expression of both YAP1 and HOG1 genes, whose products are involved in resistance to ROS, was down-regulated in fungi carrying defective NoxA, NoxB or NoxR. Our results highlight the requirement of Nox in ROS resistance and provide insights into its critical role in regulating both YAP1 and HOG1 in A.?alternata. PMID:23527595

Yang, Siwy Ling; Chung, Kuang-Ren

2013-08-01

238

Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system.  

PubMed

The phagocyte nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase was functionally reconstituted in monkey kidney COS-7 cells by transfection of essential subunits, gp91(phox), p22(phox), p47(phox), and p67(phox). COS-7 cells express the essential small guanosine 5'-triphosphatase, Rac1. Transgenic COS-phox cells were capable of arachidonic acid-induced NADPH oxidase activity up to 80% of that of human neutrophils, and of phorbol myristate acetate (PMA)-induced activity up to 20% of that of neutrophils. Expression of all 4 phox components was required for enzyme activity, and enzyme activation was associated with membrane translocation of p47(phox), p67(phox), and Rac1. Expression of p47(phox) Ser303Ala/Ser304Ala or Ser379Ala phosphorylation-deficient mutants resulted in significantly impaired NAPDH oxidase activity, compared with expression of wild-type p47(phox) or the p47(phox) Ser303Glu/Ser304Glu phosphorylation mimic, suggesting that p47(phox) phosphorylation contributes to enzyme activity in the COS system, as is the case in neutrophils. Hence, COS-phox cells should be useful as a new whole-cell model that is both capable of high-level superoxide production and readily amenable to genetic manipulation for investigation of NADPH oxidase function. PMA-elicited superoxide production in COS-phox cells was regulated by activation of protein kinase C (PKC) and Rac. Although COS-7 cells differ from human neutrophils in PKC isoform expression, transient expression of major neutrophil isoforms in COS-phox cells did not increase PMA-induced superoxide production, suggesting that endogenous isoforms were not rate limiting. Val204 in p67(phox), previously shown to be required for NADPH oxidase activity under cell-free conditions, was found to be essential for superoxide production by intact COS-phox cells, on the basis of transfection studies using a p67(phox) (Val204Ala) mutant. PMID:11929750

Price, Marianne O; McPhail, Linda C; Lambeth, J David; Han, Chang-Hoon; Knaus, Ulla G; Dinauer, Mary C

2002-04-15

239

NADPH Oxidase 1 Modulates WNT and NOTCH1 Signaling To Control the Fate of Proliferative Progenitor Cells in the Colon?  

PubMed Central

The homeostatic self-renewal of the colonic epithelium requires coordinated regulation of the canonical Wnt/?-catenin and Notch signaling pathways to control proliferation and lineage commitment of multipotent stem cells. However, the molecular mechanisms by which the Wnt/?-catenin and Notch1 pathways interplay in controlling cell proliferation and fate in the colon are poorly understood. Here we show that NADPH oxidase 1 (NOX1), a reactive oxygen species (ROS)-producing oxidase that is highly expressed in colonic epithelial cells, is a pivotal determinant of cell proliferation and fate that integrates Wnt/?-catenin and Notch1 signals. NOX1-deficient mice reveal a massive conversion of progenitor cells into postmitotic goblet cells at the cost of colonocytes due to the concerted repression of phosphatidylinositol 3-kinase (PI3K)/AKT/Wnt/?-catenin and Notch1 signaling. This conversion correlates with the following: (i) the redox-dependent activation of the dual phosphatase PTEN, causing the inactivation of the Wnt pathway effector ?-catenin, and (ii) the downregulation of Notch1 signaling that provokes derepression of mouse atonal homolog 1 (Math1) expression. We conclude that NOX1 controls the balance between goblet and absorptive cell types in the colon by coordinately modulating PI3K/AKT/Wnt/?-catenin and Notch1 signaling. This finding provides the molecular basis for the role of NOX1 in cell proliferation and postmitotic differentiation. PMID:20351171

Coant, Nicolas; Ben Mkaddem, Sanae; Pedruzzi, Eric; Guichard, Cecile; Treton, Xavier; Ducroc, Robert; Freund, Jean-Noel; Cazals-Hatem, Dominique; Bouhnik, Yoram; Woerther, Paul-Louis; Skurnik, David; Grodet, Alain; Fay, Michele; Biard, Denis; Lesuffleur, Thecla; Deffert, Christine; Moreau, Richard; Groyer, Andre; Krause, Karl-Heinz; Daniel, Fanny; Ogier-Denis, Eric

2010-01-01

240

Tempol attenuates atherosclerosis associated with metabolic syndrome via decreased vascular inflammation and NADPH-2 oxidase expression.  

PubMed

Oxidative stress is an important factor in the generation of vascular injury in atherosclerosis. Chronic administration of fructose in rodents is able to facilitate oxidative damage. In the present study we evaluated the role of Tempol, a superoxide dismutase mimetic, on the effect of high fructose intake in apolipoprotein E-deficient (ApoE-KO) mice. Rodents were fed with fructose overload (FF, 10% w/v) for 8 weeks and treated with Tempol 1 mg/kg/day the latest 4 weeks. Tempol revert the pro-oxidant effects caused by FF, diminished lipid peroxidation and impaired vascular NADPH oxidase system through the downregulation of p47phox expression in the vascular wall. Tempol inhibited the expression of vascular adhesion molecule 1 (VCAM-1) in aorta and reduced the development of atheroma plaques. Our results indicate that tempol attenuates oxidative stress by interfering with the correct assembly of Nox2 oxidase complex in the vascular wall and is able to reduce atherosclerosis. Thus tempol represents a potential therapeutic target for preventing risk factors associated with metabolic syndrome. PMID:24490696

Cannizzo, B; Quesada, I; Militello, R; Amaya, C; Miatello, R; Cruzado, M; Castro, C

2014-05-01

241

Strategies for identifying synthetic peptides to act as inhibitors of NADPH oxidases, or "all that you did and did not want to know about Nox inhibitory peptides".  

PubMed

Phagocytes utilize reactive oxygen species (ROS) to kill pathogenic microorganisms. The source of ROS is an enzymatic complex (the NADPH oxidase), comprising a membrane-associated heterodimer (flavocytochrome b (558)), consisting of subunits Nox2 and p22(phox), and four cytosolic components (p47(phox), p67(phox), p40(phox), and Rac). The primordial ROS (superoxide) is generated by the reduction of molecular oxygen by NADPH via redox centers located on Nox2. This process is activated by the translocation of the cytosolic components to the membrane and their assembly with Nox2. Membrane translocation is preceded by interactions among cytosolic components. A number of proteins structurally and functionally related to Nox2 have been discovered in many cells (the Nox family) and these have pleiotropic functions related to the production of ROS. An intense search is underway to design therapeutic means to modulate Nox-dependent overproduction of ROS, associated with diseases. Among drug candidates, a central position is held by synthetic peptides reflecting domains in oxidase components involved in NADPH oxidase assembly. Peptides, corresponding to domains in Nox2, p22(phox), p47(phox), and Rac, found to be oxidase activation inhibitory in vitro, are reviewed. Usually, peptides are inhibitory only when added preceding assembly of the complex. Although competition with intact components seems most likely, less obvious mechanisms are, sometimes, at work. The use of peptides as inhibitory drugs in vivo requires the development of methods to assure cell penetration, resistance to degradation, and avoidance of toxicity, and modest successes have been achieved. The greatest challenge remains the discovery of peptide inhibitors acting specifically on individual Nox isoforms. PMID:22562603

Dahan, Iris; Pick, Edgar

2012-07-01

242

NADPH oxidase-derived reactive oxygen species contribute to impaired cutaneous microvascular function in chronic kidney disease.  

PubMed

Oxidative stress promotes vascular dysfunction in chronic kidney disease (CKD). We utilized the cutaneous circulation to test the hypothesis that reactive oxygen species derived from NADPH oxidase and xanthine oxidase impair nitric oxide (NO)-dependent cutaneous vasodilation in CKD. Twenty subjects, 10 stage 3 and 4 patients with CKD (61 ± 4 yr; 5 men/5 women; eGFR: 39 ± 4 ml·min(-1)·1.73 m(-2)) and 10 healthy controls (55 ± 2 yr; 4 men/6 women; eGFR: >60 ml·min(-1)·1.73 m(-2)) were instrumented with 4 intradermal microdialysis fibers for the delivery of 1) Ringer solution (Control), 2) 10 ?M tempol (scavenge superoxide), 3) 100 ?M apocynin (NAD(P)H oxidase inhibition), and 4) 10 ?M allopurinol (xanthine oxidase inhibition). Skin blood flow was measured via laser-Doppler flowmetry during standardized local heating (42°C). N(g)-nitro-l-arginine methyl ester (L-NAME; 10 mM) was infused to quantify the NO-dependent portion of the response. Cutaneous vascular conductance (CVC) was calculated as a percentage of the maximum CVC achieved during sodium nitroprusside infusion at 43°C. Cutaneous vasodilation was attenuated in patients with CKD (77 ± 3 vs. 88 ± 3%, P = 0.01), but augmented with tempol and apocynin (tempol: 88 ± 2 (P = 0.03), apocynin: 91 ± 2% (P = 0.001). The NO-dependent portion of the response was reduced in patients with CKD (41 ± 4 vs. 58 ± 2%, P = 0.04), but improved with tempol and apocynin (tempol: 58 ± 3 (P = 0.03), apocynin: 58 ± 4% (P = 0.03). Inhibition of xanthine oxidase did not alter cutaneous vasodilation in either group (P > 0.05). These data suggest that NAD(P)H oxidase is a source of reactive oxygen species and contributes to microvascular dysfunction in patients with CKD. PMID:24761000

DuPont, Jennifer J; Ramick, Meghan G; Farquhar, William B; Townsend, Raymond R; Edwards, David G

2014-06-15

243

Cross talk between NADPH oxidase and autophagy in pulmonary artery endothelial cells with intrauterine persistent pulmonary hypertension.  

PubMed

Autophagy is a process for cells to degrade proteins or entire organelles to maintain a balance in the synthesis, degradation, and subsequent recycling of cellular products. Increased reactive oxygen species formation is known to induce autophagy. We previously reported that increased NADPH oxidase (NOX) activity in pulmonary artery endothelial cells (PAEC) from fetal lambs with persistent pulmonary hypertension (PPHN) contributes to impaired angiogenesis in PPHN-PAEC compared with normal PAEC. We hypothesized that increased NOX activity in PPHN-PAEC is associated with increased autophagy, which, in turn, contributes to impaired angiogenesis in PPHN-PAEC. In the present study, we detected increased autophagy in PPHN-PAEC as shown by increased ratio of the microtubule-associated protein 1 light chain (LC3)-II to LC3-I and increased percentage of green fluorescent protein-LC3 punctate positive cells. Inhibiting autophagy by 3-methyladenine, chloroquine, and beclin-1 knockdown in PPHN-PAEC has led to decreased autophagy and increased in vitro angiogenesis. Inhibition of autophagy also decreased the association between gp91(phox) and p47(phox), NOX activity, and superoxide generation. A nonspecific antioxidant N-acetylcysteine and a NOX inhibitor apocynin decreased autophagy in PPHN-PAEC. In conclusion, autophagy may contribute to impaired angiogenesis in PPHN-PAEC through increasing NOX activity. Our results suggest that, in PPHN-PAEC, a positive feedback relationship between autophagy and NOX activity may regulate angiogenesis. PMID:22245997

Teng, Ru-Jeng; Du, Jianhai; Welak, Scott; Guan, Tongju; Eis, Annie; Shi, Yang; Konduri, Girija G

2012-04-01

244

Tissue oxygen sensor function of NADPH oxidase isoforms, an unusual cytochrome aa3 and reactive oxygen species  

Microsoft Academic Search

NADPH oxidase isoforms with different gp91phox subunits as well as an unusual cytochrome aa3 with a heme a\\/a3 relationship of 9:91 are discussed as putative oxygen sensor proteins influencing gene expression and ion channel conductivity. Reactive oxygen species (ROS) are important second messengers of the oxygen sensing signal cascade determining the stability of transcription factors or the gating of ion

Torsten Porwol; Wilhelm Ehleben; Verena Brand; Helmut Acker

2001-01-01

245

Angiotensin II-Dependent Chronic Hypertension and Cardiac Hypertrophy Are Unaffected by gp91phox-Containing NADPH Oxidase  

Microsoft Academic Search

The gp91phox-containing NADPH oxidase is the major source of reactive oxygen species (ROS) in the cardiovascular system and inactivation of gp91phox has been reported to blunt hypertension and cardiac hypertrophy seen in angiotensin (Ang) II-infused animals. In the current study, we sought to determine the role of gp91phox-derived ROS on cardiovascular outcomes of chronic exposure to Ang II. The gp91phox-deficient

Rhian M. Touyz; Chantel Mercure; Ying He; Danesh Javeshghani; Guoying Yao; Glaucia E. Callera; Alvaro Yogi; Nadheige Lochard; Timothy L. Reudelhuber

2010-01-01

246

Preservation of Kidney Function with Combined Inhibition of NADPH Oxidase and Angiotensin-Converting Enzyme in Diabetic Nephropathy  

Microsoft Academic Search

Background\\/Aims: Antihypertensive therapies such as angiotensin-converting enzyme-1 inhibitors (ACEi) slow the decline in renal function seen with diabetic nephropathy, although there is still progression ultimately to end-stage renal disease. The aim of this study was to determine if there were added renoprotective benefits seen by combining ACEi with blockade of NADPH oxidase. Methods: Sprague-Dawley diabetic and non-diabetic rats were randomized

Vicki Thallas-Bonke; Melinda T. Coughlan; Leon A. Bach; Mark E. Cooper; Josephine M. Forbes

2010-01-01

247

Pharmacological inhibition of inducible nitric oxide synthase (iNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, convalesce behavior and biochemistry of hypertension induced vascular dementia in rats.  

PubMed

Cognitive disorders are likely to increase over the coming years (5-10). Vascular dementia (VaD) has heterogeneous pathology and is a challenge for clinicians. Current Alzheimer's disease drugs have had limited clinical efficacy in treating VaD and none have been approved by major regulatory authorities specifically for this disease. Role of iNOS and NADPH-oxidase has been reported in various pathological conditions but there role in hypertension (Hypt) induced VaD is still unclear. This research work investigates the salutiferous effect of aminoguanidine (AG), an iNOS inhibitor and 4'-hydroxy-3'-methoxyacetophenone (HMAP), a NADPH oxidase inhibitor in Hypt induced VaD in rats. Deoxycorticosterone acetate-salt (DOCA-S) hypertension has been used for development of VaD in rats. Morris water-maze was used for testing learning and memory. Vascular system assessment was done by testing endothelial function. Mean arterial blood pressure (MABP), oxidative stress [aortic superoxide anion, serum and brain thiobarbituric acid reactive species (TBARS) and brain glutathione (GSH)], nitric oxide levels (serum nitrite/nitrate) and cholinergic activity (brain acetyl cholinesterase activity-AChE) were also measured. DOCA-S treated rats have shown increased MABP with impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate & brain GSH levels along with increase in serum & brain TBARS, and brain AChE activity. AG as well as HMAP significantly convalesce Hypt induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that AG, an iNOS inhibitor and HMAP, a NADPH-oxidase inhibitor may be considered as potential agents for the management of Hypt induced VaD. PMID:23201648

Sharma, Bhupesh; Singh, Nirmal

2013-02-01

248

Genetic and Genomic Analysis of Rhizoctonia solani Interactions with Arabidopsis; Evidence of Resistance Mediated through NADPH Oxidases  

PubMed Central

Rhizoctonia solani is an important soil-borne necrotrophic fungal pathogen, with a broad host range and little effective resistance in crop plants. Arabidopsis is resistant to R. solani AG8 but susceptible to R. solani AG2-1. A screen of 36 Arabidopsis ecotypes and mutants affected in the auxin, camalexin, salicylic acid, abscisic acid and ethylene/jasmonic acid pathways did not reveal any variation in response to R. solani and demonstrated that resistance to AG8 was independent of these defense pathways. The Arabidopsis Affymetrix ATH1 Genome array was used to assess global gene expression changes in plants infected with AG8 and AG2-1 at seven days post-infection. While there was considerable overlap in the response, some gene families were differentially affected by AG8 or AG2-1 and included those involved in oxidative stress, cell wall associated proteins, transcription factors and heat shock protein genes. Since a substantial proportion of the gene expression changes were associated with oxidative stress responses, we analysed the role of NADPH oxidases in resistance. While single NADPH oxidase mutants had no effect, a NADPH oxidase double mutant atrbohf atrbohd resulted in an almost complete loss of resistance to AG8, suggesting that reactive oxidative species play an important role in Arabidopsis's resistance to R. solani. PMID:23451091

Foley, Rhonda C.; Gleason, Cynthia A.; Anderson, Jonathan P.; Hamann, Thorsten; Singh, Karam B.

2013-01-01

249

NAD(P)H oxidase inhibiting with apocynin improved vascular reactivity in tail-suspended hindlimb unweighting rat.  

PubMed

Recent studies suggested that reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase is of functional importance in modulating vascular tone, and we have previously detected excessive superoxide production in tail-suspended hindlimb unweighting (HU) rat cerebral and carotid arteries. HU rat was a widely used model to simulate physiological effects on the vasculature. The present study tended to investigate whether NAD(P)H oxidase inhibition with apocynin influences vasoconstriction, endothelium-dependent relaxation, and nitrite/nitrate (NOx) content in HU rat cerebral and carotid arteries. Vascular contractile and dilate responses were assessed in a myograph organ bath. NOx content in cerebral and carotid arteries was measured. We found enhanced maximal contractile response and impaired endothelium-dependent relaxation in HU rat basilar (P?NAD(P)H oxidase-derived oxidative stress mediated abnormal vasoreactivity though nitric oxide mechanism in the settings of simulated microgravity. PMID:22015782

Zhang, Ran; Ran, Hai-hong; Ma, Jin; Bai, Yun-gang; Lin, Le-jian

2012-03-01

250

Hepatocytes produce TNF-? following hypoxia-reoxygenation and liver ischemia-reperfusion in a NADPH oxidase- and c-Src-dependent manner  

PubMed Central

Cell line studies have previously demonstrated that hypoxia-reoxygenation (H/R) leads to the production of NADPH oxidase 1 and 2 (NOX1 and NOX2)-dependent reactive oxygen species (ROS) required for the activation of c-Src and NF-?B. We now extend these studies into mouse models to evaluate the contribution of hepatocytes to the NOX- and c-Src-dependent TNF-? production that follows H/R in primary hepatocytes and liver ischemia-reperfusion (I/R). In vitro, c-Src-deficient primary hepatocytes produced less ROS and TNF-? following H/R compared with controls. In vivo, c-Src-KO mice also had impaired TNF-? and NF-?B responses following partial lobar liver I/R. Studies in NOX1 and p47phox knockout primary hepatocytes demonstrated that both NOX1 and p47phox are partially required for H/R-mediated TNF-? production. To further investigate the involvement of NADPH oxidases in the production of TNF-? following liver I/R, we performed additional in vivo experiments in knockout mice deficient for NOX1, NOX2, p47phox, Rac1, and/or Rac2. Cumulatively, these results demonstrate that NOX2 and its activator subunits (p47phox and Rac) control the secretion of TNF-? by the liver following I/R. Interestingly, in the absence of Kupffer cells and NOX2, NOX1 played a dominant role in TNF-? production following hepatic I/R. However, NOX1 deletion alone had little effect on I/R-induced TNF-?. Thus Kupffer cell-derived factors and NOX2 act to suppress hepatic NOX1-dependent TNF-? production. We conclude that c-Src and NADPH oxidase components are necessary for redox-mediated production of TNF-? following liver I/R and that hepatocytes play an important role in this process. PMID:23639811

Spencer, Netanya Y.; Zhou, Weihong; Li, Qiang; Zhang, Yulong; Luo, Meihui; Yan, Ziying; Lynch, Thomas J.; Abbott, Duane; Banfi, Botond

2013-01-01

251

Oxidative stress, redox signalling and endothelial dysfunction in ageing-related neurodegenerative diseases: a role of NADPH oxidase 2.  

PubMed

Chronic oxidative stress and oxidative damage of the cerebral microvasculature and brain cells has become one of the most convincing theories in neurodegenerative pathology. Controlled oxidative metabolism and redox signalling in the central nervous system are crucial for maintaining brain function; however, excessive production of reactive oxygen species and enhanced redox signalling damage neurons. While several enzymes and metabolic processes can generate intracellular reactive oxygen species in the brain, recently an O2?-generating enzyme, NADPH oxidase 2 (Nox2), has emerged as a major source of oxidative stress in ageing-related vascular endothelial dysfunction and neurodegenerative diseases. The currently available inhibitors of Nox2 are not specific, and general antioxidant therapy is not effective in the clinic; therefore, insights into the mechanism of Nox2 activation and its signalling pathways are needed for the discovery of novel drug targets to prevent or treat these neurodegenerative diseases. This review summarizes the recent developments in understanding the mechanisms of Nox2 activation and redox-sensitive signalling pathways and biomarkers involved in the pathophysiology of the most common neurodegenerative diseases, such as ageing-related mild cognitive impairment, Alzheimer's disease and Parkinson's disease. PMID:25279404

Cahill-Smith, Sarah; Li, Jian-Mei

2014-09-01

252

Tea polyphenols alleviate high fat and high glucose-induced endothelial hyperpermeability by attenuating ROS production via NADPH oxidase pathway  

PubMed Central

Background Hyperglycemia-induced endothelial hyperpermeability is crucial to cardiovascular disorders and macro-vascular complications in diabetes mellitus. The objective of this study is to investigate the effects of green tea polyphenols (GTPs) on endothelial hyperpermeability and the role of nicotinamide adenine dinucleotide phosphate (NADPH) pathway. Methods Male Wistar rats fed on a high fat diet (HF) were treated with GTPs (0, 0.8, 1.6, 3.2 g/L in drinking water) for 26 weeks. Bovine aortic endothelial cells (BAECs) were treated with high glucose (HG, 33 mmol/L) and GTPs (0.0, 0.4, or 4 ?g/mL) for 24 hours in vitro. The endothelial permeabilities in rat aorta and monolayer BAECs were measured by Evans blue injection method and efflux of fluorescein isothiocyanate (FITC)-dextran, respectively. The reactive oxygen species (ROS) levels in rat aorta and monolayer BAECs were measured by dihydroethidium (DHE) and 2?, 7?-dichloro-fluorescein diacetate (DCFH-DA) fluorescent probe, respectively. Protein levels of NADPH oxidase subunits were determined by Western-blot. Results HF diet-fed increased the endothelial permeability and ROS levels in rat aorta while HG treatments increased the endothelial permeability and ROS levels in cultured BAECs. Co-treatment with GTPs alleviated those changes both in vivo and in vitro. In in vitro studies, GTPs treatments protected against the HG-induced over-expressions of p22phox and p67phox. Diphenylene iodonium chloride (DPI), an inhibitor of NADPH oxidase, alleviated the hyperpermeability induced by HG. Conclusions GTPs could alleviate endothelial hyperpermeabilities in HF diet-fed rat aorta and in HG treated BAECs. The decrease of ROS production resulting from down-regulation of NADPH oxidase contributed to the alleviation of endothelial hyperpermeability. PMID:24580748

2014-01-01

253

Emerging role of oxidative stress in metabolic syndrome and cardiovascular diseases: important role of Rac/NADPH oxidase.  

PubMed

'Oxidative stress' is a term defining states of elevated reactive oxygen species (ROS) levels. Normally, ROS control several physiological processes, such as host defence, biosynthesis of hormones, fertilization and cellular signalling. However, oxidative stress has been involved in different pathologies, including metabolic syndrome and numerous cardiovascular diseases. A major source of ROS involved in both metabolic syndrome and cardiovascular pathophysiology is the NADPH oxidase (NOX) family of enzymes. NOX is a multi-component enzyme complex that consists of membrane-bound cytochrome b-558, which is a heterodimer of gp91phox and p22phox, cytosolic regulatory subunits p47phox and p67phox, and the small GTP-binding protein Rac1. Rac1 plays many important biological functions in cells, but perhaps the most unique function of Rac1 is its ability to bind and activate the NOX complex. Furthermore, Rac1 has been reported to be a key regulator of oxidative stress through its co-regulatory effects on both nitric oxide (NO) synthase and NOX. Therefore, the main goal of this review is to give a brief outline about the important role of the Rac1-NOX axis in the pathophysiology of both metabolic syndrome and cardiovascular disease. PMID:24037780

Elnakish, Mohammad T; Hassanain, Hamdy H; Janssen, Paul M; Angelos, Mark G; Khan, Mahmood

2013-11-01

254

NADPH oxidase NOX4 supports renal tumorigenesis by promoting the expression and nuclear accumulation of HIF2?.  

PubMed

Most sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor-2? (HIF2?), a master transcriptional regulator of genes that drive diverse processes, including angiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2? expression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions of NOX4 to RCC have not been evaluated directly. Here, we report that NOX4 silencing in VHL-deficient RCC cells abrogates cell branching, invasion, colony formation, and growth in a murine xenograft model RCC. These alterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression of manganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficient to block nuclear accumulation of HIF2? in RCC cells. Our results offer direct evidence that NOX4 is critical for renal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cells are genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade. PMID:24755467

Gregg, Jennifer L; Turner, Robert M; Chang, Guimin; Joshi, Disha; Zhan, Ye; Chen, Li; Maranchie, Jodi K

2014-07-01

255

Reactive oxygen species (ROS) from NADPH and xanthine oxidase modulate the cutaneous local heating response in healthy humans  

PubMed Central

Local cutaneous heating produces vasodilation that is largely nitric oxide (NO) dependent. We showed that angiotensin II (ANG II) attenuates this by an ANG II receptor, type 1 (AT1R)-dependent mechanism that is reversible with the antioxidant ascorbate, indicating oxidative stress. Reactive oxygen species (ROS) produced by ANG II employ NADPH and xanthine oxidase pathways. To determine whether these mechanisms pertain to skin, we measured cutaneous local heating with 10 ?M ANG II, using apocynin to inhibit NADPH oxidase and allopurinol to inhibit xanthine oxidase. We also inhibited superoxide with tempol, and H2O2 with ebselen. We heated the skin of the calf in 8 healthy volunteers (24.5–29.9 yr old) to 42°C and measured local blood flow to assess the percentage of maximum cutaneous vascular conductance. We remeasured while perfusing allopurinol, apocynin, ebselen, and tempol through individual microdialysis catheters. This was then repeated with ANG II combined with antioxidant drugs. tempol and apocynin alone had no effect on the heat response. Allopurinol enhanced the entire response (125% of heat alone), while ebselen suppressed the heat plateau (76% of heat alone). ANG II alone caused significant attenuation of the entire heat response (52%). When added to ANG II, Allopurinol partially reversed the ANG II attenuation. Heat with ebselen and ANG II were similar to heat and ANG II; ebselen only partially reversed the ANG II attenuation. Apocynin and tempol each partially reversed the attenuation caused by ANG II. This suggests that ROS, produced by ANG II via NADPH and xanthine oxidase pathways, modulates the response of skin to the application of heat, and thus contributes to the control of local cutaneous blood flow. PMID:21436462

Bamji, Natasha; Clarke, Debbie; Ocon, Anthony J.; Stewart, Julian M.

2011-01-01

256

NADPH oxidase-2 inhibition restores contractility and intracellular calcium handling and reduces arrhythmogenicity in dystrophic cardiomyopathy.  

PubMed

Duchenne muscular dystrophy may affect cardiac muscle, producing a dystrophic cardiomyopathy in humans and the mdx mouse. We tested the hypothesis that oxidative stress participates in disrupting calcium handling and contractility in the mdx mouse with established cardiomyopathy. We found increased expression (fivefold) of the NADPH oxidase (NOX) 2 in the mdx hearts compared with wild type, along with increased superoxide production. Next, we tested the impact of NOX2 inhibition on contractility and calcium handling in isolated cardiomyocytes. Contractility was decreased in mdx myocytes compared with wild type, and this was restored toward normal by pretreating with apocynin. In addition, the amplitude of evoked intracellular Ca(2+) concentration transients that was diminished in mdx myocytes was also restored with NOX2 inhibition. Total sarcoplasmic reticulum (SR) Ca(2+) content was reduced in mdx hearts and normalized by apocynin treatment. Additionally, NOX2 inhibition decreased the production of spontaneous diastolic calcium release events and decreased the SR calcium leak in mdx myocytes. In addition, nitric oxide (NO) synthase 1 (NOS-1) expression was increased eightfold in mdx hearts compared with wild type. Nevertheless, cardiac NO production was reduced. To test whether this paradox implied NOS-1 uncoupling, we treated cardiac myocytes with exogenous tetrahydrobioterin, along with the NOX inhibitor VAS2870. These agents restored NO production and phospholamban phosphorylation in mdx toward normal. Together, these results demonstrate that, in mdx hearts, NOX2 inhibition improves the SR calcium handling and contractility, partially by recoupling NOS-1. These findings reveal a new layer of nitroso-redox imbalance in dystrophic cardiomyopathy. PMID:25015966

Gonzalez, Daniel R; Treuer, Adriana V; Lamirault, Guillaume; Mayo, Vera; Cao, Yenong; Dulce, Raul A; Hare, Joshua M

2014-09-01

257

Phagocyte-like NADPH oxidase generates ROS in INS 832/13 cells and rat islets: role of protein prenylation  

PubMed Central

Recent evidence suggests that an acute increase in the generation of phagocyte-like NADPH-oxidase (Nox)-mediated reactive oxygen species (ROS) may be necessary for glucose-stimulated insulin secretion. Using rat islets and INS 832/13 cells, we tested the hypothesis that activation of specific G proteins is necessary for nutrient-mediated intracellular generation of ROS. Stimulation of ?-cells with glucose or a mixture of mitochondrial fuels (mono-methylsuccinate plus ?-ketoisocaproic acid) markedly elevated intracellular accumulation of ROS, which was attenuated by selective inhibitors of Nox (e.g., apocynin or diphenyleneiodonium chloride) or short interfering RNA-mediated knockdown of p47phox, one of the subunits of Nox. Selective inhibitors of protein prenylation (FTI-277 or GGTI-2147) markedly inhibited nutrient-induced ROS generation, suggesting that activation of one (or more) prenylated small G proteins and/or ?-subunits of trimeric G proteins is involved in this signaling axis. Depletion of endogenous GTP levels with mycophenolic acid significantly reduced glucose-induced activation of Rac1 and ROS generation in these cells. Other immunosuppressants, like cyclosporine A or rapamycin, which do not deplete endogenous GTP levels, failed to affect glucose-induced ROS generation, suggesting that endogenous GTP is necessary for glucose-induced Nox activation and ROS generation. Treatment of INS 832/13 cells or rat islets with pertussis toxin (Ptx), which ADP ribosylates and inhibits inhibitory class of trimeric G proteins (i.e., Gi or Go), significantly attenuated glucose-induced ROS generation in these cells, implicating activation of a Ptx-sensitive G protein in these signaling cascade. Together, our findings suggest a prenylated Ptx-sensitive signaling step couples Rac1 activation in the signaling steps necessary for glucose-mediated generation of ROS in the pancreatic ?-cells. PMID:21228337

Syed, Ismail; Kyathanahalli, Chandrashekara N.

2011-01-01

258

NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells  

SciTech Connect

Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to chronic inflammation and the development of vascular diseases. Because cell adhesion molecules (CAMs) of the cerebrovascular endothelium regulate infiltration of inflammatory cells into the brain, we have explored the molecular mechanisms by which ortho-substituted polychlorinated biphenyls (PCBs), such as PCB153, can upregulate CAMs in brain endothelial cells. Exposure to PCB153 increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as elevated adhesion of leukocytes to brain endothelial cells. These effects were impeded by inhibitors of EGFR, JAKs, or Src activity. In addition, pharmacological inhibition of NADPH oxidase or disruption of lipid rafts by cholesterol depleting agents blocked PCB153-induced phosphorylation of JAK and Src kinases and upregulation of CAMs. In contrast, silencing of caveolin-1 by siRNA interference did not affect upregulation of ICAM-1 and VCAM-1 in brain endothelial cells stimulated by PCB153. Results of the present study indicate that lipid raft-dependent NADPH oxidase/JAK/EGFR signaling mechanisms regulate the expression of CAMs in brain endothelial cells and adhesion of leukocytes to endothelial monolayers. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCB-induced cerebrovascular disorders and neurotoxic effects in the CNS.

Eum, Sung Yong [Molecular Neuroscience and Vascular Biology Laboratory, Department of Neurosurgery, University of Kentucky, Lexington, KY 40536 (United States)], E-mail: sungyong.eum@uky.edu; Andras, Ibolya [Molecular Neuroscience and Vascular Biology Laboratory, Department of Neurosurgery, University of Kentucky, Lexington, KY 40536 (United States); Hennig, Bernhard [College of Agriculture, University of Kentucky, Lexington, KY 40536 (United States); Toborek, Michal [Molecular Neuroscience and Vascular Biology Laboratory, Department of Neurosurgery, University of Kentucky, Lexington, KY 40536 (United States)

2009-10-15

259

A novel pathway of NADPH oxidase/vascular peroxidase 1 in mediating oxidative injury following ischemia-reperfusion.  

PubMed

Vascular peroxidase 1 (VPO1) can utilize reactive oxygen species (ROS) generated from NADPH oxidase (NOX) to catalyze peroxidative reactions. This study was performed to identify a novel pathway of NOX/VPO1 in mediating the oxidative injury following myocardial ischemia reperfusion (IR). In a rat model of myocardial IR, the infarct size, serum creatine kinase (CK) activity, apoptosis, NOX activity, NOX2 and VPO1 expression were measured. In a cell (rat heart-derived H9c2 cells) model of hypoxia/reoxygenation (HR), the apoptosis, NOX activity, NOX2 and VPO1 expression, and H(2)O(2) and HOCl levels were examined. In vivo, IR caused 54.8 ± 1.7 % infarct size in myocardium accompanied by elevated activities of CK, caspase-3 and NOX, up-regulated VPO1 expression and high numbers of myocardial apoptotic cells; these effects were attenuated by pretreatment with the inhibitor of NOX. In vitro, inhibition of NOX or silencing of NOX2 or VPO1 expression significantly suppressed HR-induced cellular apoptosis concomitantly with decreased HOCl production. Inhibition of NOX or silencing of NOX2 led to a decrease in H(2)O(2) production accompanied by a decrease in VPO1 expression and HOCl production. However, silencing of VPO1 expression did not affect NOX2 expression and H(2)O(2) production. H(2)O(2)-induced VPO1 expression was partially reversed by JNK or p38 MAPK inhibitor. Our results demonstrate a novel pathway of NOX2/VPO1 in myocardium, where VPO1 coordinates with NOX2 and amplifies the role of NOX-derived ROS in oxidative injury following IR. PMID:22476986

Zhang, Yi-Shuai; He, Lan; Liu, Bin; Li, Nian-Sheng; Luo, Xiu-Ju; Hu, Chang-Ping; Ma, Qi-Lin; Zhang, Guo-Gang; Li, Yuan-Jian; Peng, Jun

2012-05-01

260

The Crystal Structure of the Plant Small GTPase OsRac1 Reveals Its Mode of Binding to NADPH Oxidase.  

PubMed

Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(?,?-imido)triphosphate at 1.9 Ĺ resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins. PMID:25128531

Kosami, Ken-Ichi; Ohki, Izuru; Nagano, Minoru; Furuita, Kyoko; Sugiki, Toshihiko; Kawano, Yoji; Kawasaki, Tsutomu; Fujiwara, Toshimichi; Nakagawa, Atsushi; Shimamoto, Ko; Kojima, Chojiro

2014-10-10

261

Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases.  

PubMed

Activation of microglial NADPH oxidase (NOX2) plays a critical role in mediating neuroinflammation, which is closely linked with the pathogenesis of a variety of neurodegenerative diseases, including Parkinson's disease (PD). The inhibition of NOX2-generated superoxide has become an effective strategy for developing disease-modifying therapies for PD. However, the lack of specific and potent NOX2 inhibitors has hampered the progress of this approach. Diphenyleneiodonium (DPI) is a widely used, long-acting NOX2 inhibitor. However, due to its non-specificity for NOX2 and high cytotoxicity at standard doses (µM), DPI has been precluded from human studies. In this study, using ultra-low doses of DPI, we aimed to: (1) investigate whether these problems could be circumvented and (2) determine whether ultra-low doses of DPI were able to preserve its utility as a potent NOX2 inhibitor. We found that DPI at subpicomolar concentrations (10(-14) and 10(-13) M) displays no toxicity in primary midbrain neuron-glia cultures. More importantly, we observed that subpicomolar DPI inhibited phorbol myristate acetate (PMA)-induced activation of NOX2. The same concentrations of DPI did not inhibit the activities of a series of flavoprotein-containing enzymes. Furthermore, potent neuroprotective efficacy was demonstrated in a post-treatment study. When subpicomolar DPI was added to neuron-glia cultures pretreated with lipopolysaccharide, 1-methyl-4-phenylpyridinium or rotenone, it potently protected the dopaminergic neurons. In summary, DPI's unique combination of high specificity toward NOX2, low cytotoxicity and potent neuroprotective efficacy in post-treatment regimens suggests that subpicomolar DPI may be an ideal candidate for further animal studies and potential clinical trials. GLIA 2014;62:2034-2043. PMID:25043383

Wang, Qingshan; Chu, Chun-Hsien; Oyarzabal, Esteban; Jiang, Lulu; Chen, Shih-Heng; Wilson, Belinda; Qian, Li; Hong, Jau-Shyong

2014-12-01

262

A plant homolog of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca2+ binding motifs.  

PubMed Central

Rapid generation of O2- and H2O2, which is reminiscent of the oxidative burst in neutrophils, is a central component of the resistance response of plants to pathogen challenge. Here, we report that the Arabidopsis rbohA (for respiratory burst oxidase homolog A) gene encodes a putative 108-kD protein, with a C-terminal region that shows pronounced similarity to the 69-kD apoprotein of the gp91phox subunit of the neutrophil respiratory burst NADPH oxidase. The RbohA protein has a large hydrophilic N-terminal domain that is not present in gp91phox. This domain contains two Ca2+ binding EF hand motifs and has extended similarity to the human RanGTPase-activating protein 1. rbohA, which is a member of a divergent gene family, generates transcripts of 3.6 and 4.0 kb that differ only in their polyadenylation sites. rbohA transcripts are most abundant in roots, with weaker expression in aerial organs and seedlings. Antibodies raised against a peptide near the RbohA C terminus detected a 105-kD protein that, unlike gp91phox, does not appear to be highly glycosylated. Cell fractionation, two-phase partitioning, and detergent extraction indicate that RbohA is an intrinsic plasma membrane protein. We propose that plants have a plasma membrane enzyme similar to the neutrophil NADPH oxidase but with novel potential regulatory mechanisms for Ca2+ and G protein stimulation of O2- and H2O2 production at the cell surface. PMID:9490748

Keller, T; Damude, H G; Werner, D; Doerner, P; Dixon, R A; Lamb, C

1998-01-01

263

Ionizing irradiation induces apoptotic damage of salivary gland acinar cells via NADPH oxidase 1-dependent superoxide generation  

SciTech Connect

Reactive oxygen species (ROS) have important roles in various physiological processes. Recently, several novel homologues of the phagocytic NADPH oxidase have been discovered and this protein family is now designated as the Nox family. We investigated the involvement of Nox family proteins in ionizing irradiation-induced ROS generation and impairment in immortalized salivary gland acinar cells (NS-SV-AC), which are radiosensitive, and immortalized ductal cells (NS-SV-DC), which are radioresistant. Nox1-mRNA was upregulated by {gamma}-ray irradiation in NS-SV-AC, and the ROS level in NS-SV-AC was increased to approximately threefold of the control level after 10 Gy irradiation. The increase of ROS level in NS-SV-AC was suppressed by Nox1-siRNA-transfection. In parallel with the suppression of ROS generation and Nox1-mRNA expression by Nox1-siRNA, ionizing irradiation-induced apoptosis was strongly decreased in Nox1-siRNA-transfected NS-SV-AC. There were no large differences in total SOD or catalase activities between NS-SV-AC and NS-SV-DC although the post-irradiation ROS level in NS-SV-AC was higher than that in NS-SV-DC. In conclusion, these results indicate that Nox1 plays a crucial role in irradiation-induced ROS generation and ROS-associated impairment of salivary gland cells and that Nox1 gene may be targeted for preservation of the salivary gland function from radiation-induced impairment.

Tateishi, Yoshihisa [Department of Oral and Maxillofacial Surgery, Kochi Medical School, Kochi University, Kohasu, Oko-cho, Nankoku-city, Kochi 783-8505 (Japan)], E-mail: tateishi@kochi-u.ac.jp; Sasabe, Eri; Ueta, Eisaku; Yamamoto, Tetsuya [Department of Oral and Maxillofacial Surgery, Kochi Medical School, Kochi University, Kohasu, Oko-cho, Nankoku-city, Kochi 783-8505 (Japan)

2008-02-08

264

Receptor Signaling Lymphocyte-activation Molecule Family 1 (Slamf1) Regulates Membrane Fusion and NADPH Oxidase 2 (NOX2) Activity by Recruiting a Beclin-1/Vps34/Ultraviolet Radiation Resistance-associated Gene (UVRAG) Complex*  

PubMed Central

Phagocytosis is a pivotal process by which macrophages eliminate microorganisms upon recognition by pathogen sensors. Surprisingly, the self-ligand cell surface receptor Slamf1 functions not only as a co-stimulatory molecule but also as a microbial sensor of several Gram-negative bacteria. Upon entering the phagosome of macrophages Slamf1 induces production of phosphatidylinositol 3-phosphate, which positively regulates the activity of the NOX2 enzyme and phagolysosomal maturation. Here, we report that in Escherichia coli-containing phagosomes of mouse macrophages, Slamf1 interacts with the class III PI3K Vps34 in a complex with Beclin-1 and UVRAG. Upon phagocytosis of bacteria the NOX2 activity was reduced in macrophages isolated from Beclin-1+/? mice compared with wild-type mice. This Slamf1/Beclin-1/Vps34/UVRAG protein complex is formed in intracellular membrane compartments as it is found without inducing phagocytosis in macrophages, human chronic lymphocytic leukemia cells, and transfectant HEK293 cells. Elimination of its cytoplasmic tail abolished the interaction of Slamf1 with the complex, but deletion or mutation of the two ITAM motifs did not. Both the BD and CCD domains of Beclin-1 were required for efficient binding to Slamf1. Because Slamf1 did not interact with Atg14L or Rubicon, which can also form a complex with Vps34 and Beclin-1, we conclude that Slamf1 recruits a subset of Vps34-associated proteins, which is involved in membrane fusion and NOX2 regulation. PMID:22493499

Ma, Chunyan; Wang, Ninghai; Detre, Cynthia; Wang, Guoxing; O'Keeffe, Michael; Terhorst, Cox

2012-01-01

265

Endothelial NADPH oxidase 4 mediates vascular endothelial growth factor receptor 2-induced intravitreal neovascularization in a rat model of retinopathy of prematurity  

PubMed Central

Purpose NADPH oxidase–generated reactive oxygen species (ROS) are implicated in angiogenesis. Isoforms of NADPH oxidase NOX1, NOX2, and NOX4 are reported to be expressed in endothelial cells (ECs). Of these, NOX1 and NOX2 have been reported to contribute to intravitreal neovascularization (IVNV) in oxygen-induced retinopathy (OIR) models. In this study, we tested the hypothesis that the isoform NOX4 in ECs contributed to vascular endothelial growth factor (VEGF)–induced angiogenesis and IVNV. Methods Isoforms of NADPH oxidase mRNA were measured in several types of cultured vascular ECs: human retinal microvascular ECs (hRMVECs), choroidal ECs (CECs), and human umbilical vascular ECs (HUVECs) using real-time PCR. Newborn rat pups and dams were placed into an OIR model that cycled oxygen concentration between 50% and 10% every 24 h for 14 days, and then were placed in room air (RA) for an additional 4 days (rat OIR model). NOX4 expression in retinal lysates from the RA–raised pups at postnatal day 0 (P0), P14, and P18 was determined with western blots. STAT3 activation was determined as the ratio of phosphorylated STAT3 to total STAT3 with western blot analysis of retinal lysates from pups raised in RA or from the rat OIR model at P18. Semiquantitative assessment of the density of NOX4 colabeling with lectin-stained retinal ECs was determined by immunolabeling of retinal cryosections from P18 pups in OIR or in RA. In hRMVECs transfected with NOX4 siRNA and treated with VEGF or control, 1) ROS generation was measured using the 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester fluorescence assay and 2) phosphorylated VEGF receptor 2 and STAT3, and total VEGFR2 and STAT3 were measured in western blot analyses. VEGF-stimulated hRMVEC proliferation was measured following transfection with NOX4 siRNA or STAT3 siRNA, or respective controls. Results NOX4 was the most prevalent isoform of NADPH oxidase in vascular ECs. NOX4 expression in retinal lysates was significantly decreased during development in RA. Compared to RA, the expression of retinal NOX4 increased at P18. At p18 OIR, semiquantitative assessment of the density of lectin and NOX4 colabeling in retinal vascular ECs was greater in retinal cryosections and activated STAT3 was greater in retinal lysates when compared to the RA-raised pups. In cultured hRMVECs, knockdown of NOX4 by siRNA transfection inhibited VEGF-induced ROS generation. VEGF induced a physical interaction of phosphorylated-VEGFR2 and NOX4. Knockdown of NOX4: 1) reduced VEGFR2 activation but did not abolish it and 2) abolished STAT3 activation in response to VEGF. Knockdown of either NOX4 or STAT3 inhibited VEGF-induced EC proliferation. Conclusions Our data suggest that in a model representative of human retinopathy of prematurity, NOX4 was increased at a time point when IVNV developed. VEGF-activated NOX4 led to an interaction between VEGF-activated VEGFR2 and NOX4 that mediated EC proliferation via activation of STAT3. Altogether, our results suggest that NOX4 may regulate VEGFR2-mediated IVNV through activated STAT3. PMID:24623966

Wang, Haibo; Yang, Zhihong; Jiang, Yanchao

2014-01-01

266

Genipin induces cyclooxygenase-2 expression via NADPH oxidase, MAPKs, AP-1, and NF-?B in RAW 264.7 cells.  

PubMed

Genipin is a compound found in gardenia fruit extract with diverse pharmacological activities. However, the mechanism underlying genipin-induced cyclooxygenase-2 (COX-2) expression remains unknown. In this study, we investigated the effects of genipin on COX-2 expression and determined that exposure to genipin dose-dependently enhanced the production of prostaglandin E2 (PGE2), a major COX-2 metabolite, in RAW 264.7 cells. These effects were mediated by genipin-induced activation of the COX-2 promoter, as well as AP-1 and NF-?B luciferase constructs. Phosphatidylinositol-3-kinase/Akt and MAPKs were also significantly activated by genipin, and Akt and MAPKs inhibitors (PD98059, SB20358, SP600125, and LY294002) inhibited genipin-induced COX-2 expression. Moreover, genipin increased production of the ROS and the ROS-producing NAPDH-oxidase (NOX) family oxidases, NOX2 and NOX3. Inhibition of NADPH with diphenyleneiodonium attenuated ROS production, COX-2 expression and NF-?B and AP-1 activation. These results suggest that the molecular mechanism mediating ROS-dependent COX-2 up-regulation and PGE2 production by genipin involves activation of Akt, MAPKs and AP-1/NF-?B. PMID:24296130

Khanal, Tilak; Kim, Hyung Gyun; Do, Minh Truong; Choi, Jae Ho; Chung, Young Chul; Kim, Hee Suk; Park, Youn-Joon; Jeong, Tae Cheon; Jeong, Hye Gwang

2014-02-01

267

Essential Role of NADPH Oxidase-Dependent Reactive Oxygen Species Generation in Regulating MicroRNA-21 Expression and Function in Prostate Cancer  

PubMed Central

Abstract Aims: Oncogenic microRNAs (miRs) promote tumor growth and invasiveness. One of these, miR-21, contributes to carcinogenesis in prostate and other cancers. In the present study, we tested the hypothesis that NADPH oxidase-dependent reactive oxygen species (ROS) regulate the expression and function of miR-21 and its target proteins, maspin and programmed cell death 4 (PDCD4), in prostate cancer cells. Results: The highly aggressive androgen receptor negative PC-3M-MM2 prostate cancer cells demonstrated high expression of miR-21 and p47phox (an essential subunit of NADPH oxidase). Using loss-of-function strategy, we showed that transfection of PC-3M-MM2 cells with anti-miR-21- and p47phox siRNA (si-p47phox) led to reduced expression of miR-21 with concurrent increase in maspin and PDCD4, and decreased the invasiveness of the cells. Tail-vein injections of anti-miR-21- and si-p47phox-transfected PC-3M-MM2 cells in severe combined immunodeficient mice reduced lung metastases. Clinical samples from patients with advanced prostate cancer expressed high levels of miR-21 and p47phox, and low expression of maspin and PDCD4. Finally, ROS activated Akt in these cells, the inhibition of which reduced miR-21 expression. Innovation: The levels of NADPH oxidase-derived ROS are high in prostate cancer cells, which have been shown to be involved in their growth and migration. This study demonstrates that ROS produced by this pathway is essential for the expression and function of an onco-miR, miR-21, in androgen receptor-negative prostate cancer cells. Conclusion: These data demonstrate that miR-21 is an important target of ROS, which contributes to the highly invasive and metastatic phenotype of prostate cancer cells. Antioxid. Redox Signal. 19, 1863–1876. PMID:23682737

Jajoo, Sarvesh; Mukherjea, Debashree; Kaur, Tejbeer; Sheehan, Kelly E.; Sheth, Sandeep; Borse, Vikrant; Rybak, Leonard P.

2013-01-01

268

Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate.  

PubMed

Phagocytic leukocytes contain an activatable NADPH:O2 oxidoreductase. Components of this enzyme system include cytochrome b558, and three soluble oxidase components (SOC I, SOC II, and SOC III) found in the cytosol of resting cells. Previously, we found that SOC II copurifies with, and is probably identical to, a 47-kDa substrate of protein kinase C. In the present study we investigated the change in location of several of these oxidase components after activation of intact neutrophils with phorbol myristate acetate (PMA) and separation of subcellular fraction on sucrose density gradients. On Western blots with fractions of resting cells, the alpha subunit of cytochrome b558 was detected with a monoclonal antibody as a doublet of Mr 22,000 and 24,000 in the specific granules and as a single band of Mr 24,000 in the plasma membrane. PMA induced an increase of cytochrome b558 in the plasma membrane, including the Mr 22,000 band. PMA also induced translocation of the 47-kDa protein from the cytosol to the membrane fraction, as revealed by in vitro phosphorylation experiments. When NADPH oxidase activity was determined in a cell-free system in the presence of sodium dodecyl sulfate and GTP with plasma membranes from resting cells, cytosol from PMA-treated cells was deficient compared with cytosol from resting cells. This deficiency could be partially restored by the addition of SOC I. Concomitantly, SOC I activity appeared in the plasma membranes of PMA-treated cells. These studies support the hypothesis that PMA stimulation of neutrophils results in assembly of oxidase components from the cytosol and the specific granules in the plasma membrane with subsequent expression of NADPH oxidase activity. PMID:2153119

Ambruso, D R; Bolscher, B G; Stokman, P M; Verhoeven, A J; Roos, D

1990-01-15

269

NAD(P)H oxidase and pro-inflammatory response during maximal exercise: role of C242T polymorphism of the P22PHOX subunit.  

PubMed

Intense exercise induces a pro-inflammatory status through a mechanism involving the NAD(P)H oxidase system. We focused our attention on p22phox, a subunit of the NAD(P)H oxidase, and on its allelic polymorphism C242T, which is known to affect the functional activity of the enzyme. We investigated whether the p22phox C242T variants exhibit systemic effects in healthy subjects by analyzing the proinflammatory and cardiocirculatory responses to physical exercise in endurance athletes. The group of study consisted of 97 long distance runners, 37 +/- 4.4 yrs of age, with similar training history. The subjects underwent a maximal stress test during which both inflammatory and cardiopulmonary parameters were monitored. Our results demonstrate that T allele deeply influences the neutrophil activation in response to intense exercise, since T carriers were characterized by significantly lower release of myeloperoxidase (MPO), a classical leukocyte derived pro-inflammatory cytokine. In addition, the presence of T allele was associated with a higher cardiopulmonary efficiency as evidenced by a significantly lower Heart Rate (HR) at the peak of exercise and, when a dominant model was assumed, by a higher maximal oxygen uptake (VO2 max). On the other hand, no effects of 242T mutation on the plasmatic total antioxidant capacity (TAC) and on the cortisol responses to the physical exercise were detected. In conclusion, our data support a systemic role for p22phox C242T polymorphism that, modifying the intensity of the inflammatory response, can influence the cardiovascular adaptations elicited by aerobic training. These results contribute to support the hypothesis of a systemic effect for the C242T polymorphism and of its possible functional rebound in healthy subjects. PMID:20378006

Izzicupo, P; Di Valerio, V; D' Amico, M A; Di Mauro, M; Pennelli, A; Falone, S; Alberti, G; Amicarelli, F; Miscia, S; Gallina, S; Di Baldassarre, A

2010-01-01

270

Transcriptional regulation of NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-{kappa}B in human aortic smooth muscle cells  

SciTech Connect

Inflammation-induced changes in the activity and expression of NADPH oxidases (Nox) play a key role in atherogenesis. The molecular mechanisms of Nox regulation are scantily elucidated. Since nuclear factor-{kappa}B (NF-{kappa}B) controls the expression of many genes associated to inflammation-related diseases, in this study we have investigated the role of NF-{kappa}B signaling in the regulation of Nox1 and Nox4 transcription in human aortic smooth muscle cells (SMCs). Cultured cells were exposed to tumor necrosis factor-{alpha} (TNF{alpha}), a potent inducer of both Nox and NF-{kappa}B, up to 24 h. Lucigenin-enhanced chemiluminescence and dichlorofluorescein assays, real-time polymerase chain reaction, and Western blot analysis showed that inhibition of NF-{kappa}B pathway reduced significantly the TNF{alpha}-dependent up-regulation of Nox-derived reactive oxygen species production, Nox1 and Nox4 expression. In silico analysis indicated the existence of typical NF-{kappa}B elements in the promoters of Nox1 and Nox4. Transient overexpression of p65/NF-{kappa}B significantly increased the promoter activities of both isoforms. Physical interaction of p65/NF-{kappa}B proteins with the predicted sites was demonstrated by chromatin immunoprecipitation assay. These findings demonstrate that NF-{kappa}B is an essential regulator of Nox1- and Nox4-containing NADPH oxidase in SMCs. Elucidation of the complex relationships between NF-{kappa}B and Nox enzymes may lead to a novel pharmacological strategy to reduce both inflammation and oxidative stress in atherosclerosis and its associated complications.

Manea, Adrian, E-mail: adrian.manea@icbp.ro [Institute of Cellular Biology and Pathology 'Nicolae Simionescu', 8, B.P. Hasdeu Street, Bucharest, P.O. Box 35-14 (Romania)] [Institute of Cellular Biology and Pathology 'Nicolae Simionescu', 8, B.P. Hasdeu Street, Bucharest, P.O. Box 35-14 (Romania); Tanase, Laurentia I.; Raicu, Monica; Simionescu, Maya [Institute of Cellular Biology and Pathology 'Nicolae Simionescu', 8, B.P. Hasdeu Street, Bucharest, P.O. Box 35-14 (Romania)] [Institute of Cellular Biology and Pathology 'Nicolae Simionescu', 8, B.P. Hasdeu Street, Bucharest, P.O. Box 35-14 (Romania)

2010-06-11

271

NADPH-oxidase and a Hydrogen Peroxide-Sensitive K+ Channel May Function as an Oxygen Sensor Complex in Airway Chemoreceptors and Small Cell Lung Carcinoma Cell Lines  

Microsoft Academic Search

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage

Dashou Wang; Charlotte Youngson; Veronica Wong; Herman Yeger; Mary C. Dinauer; Eleazar Vega-Saenz de Miera; Bernardo Rudy; Ernest Cutz

1996-01-01

272

Phycobiliprotein C-phycocyanin from Spirulina platensis is powerfully responsible for reducing oxidative stress and NADPH oxidase expression induced by an atherogenic diet in hamsters.  

PubMed

The effects of spirulina and its chromophore phycocyanin, both without bound Se or selenium-enriched, were studied on plasma cholesterol, early atherosclerosis, cardiac production of superoxide anions, and NAD(P)H oxidase expression in hamsters. Forty hamsters were divided into 5 groups of 8 and fed an atherogenic diet for 12 weeks. They received by gavage either 7.14 mL/(kg day) phycocyanin (PC), Se-rich phycocyanin (SePC), spirulina (SP) or Se-rich spirulina (SeSP) in water, or water as control. SeSP and SePC supplied 0.4 microg of Se per 100 g body weight. Plasma cholesterol and non-HDL cholesterol concentrations were lower in group consuming SePC. HDL-cholesterol was never affected. SePC significantly increased plasma antioxidant capacity by 42% compared with controls. A sparing effect in liver glutathione peroxidase (87% on average) and superoxide dismutase (56% on average) activity was observed for all the groups compared to controls. Aortic fatty streak area was significantly reduced in the experimental groups, especially by PC (82%) and SePC (85%). Cardiac production of superoxide anion significantly decreased by approximately 46-76% in the four experimental groups and especially in SePC group (76%). The expression of p22phox subunit of NAD(P)H oxidase decreased by 34% after consumption of SePC. The results indicate that chronic consumption of Se-rich spirulina phycocyanin powerfully prevents the development of atherosclerosis. The underlying mechanism is related mainly to inhibiting pro-oxidant factors and at a lesser extent improving the serum lipid profile. PMID:17696484

Riss, Jérome; Décordé, Kelly; Sutra, Thibault; Delage, Martine; Baccou, Jean-Claude; Jouy, Nicolas; Brune, Jean-Pierre; Oréal, Henri; Cristol, Jean-Paul; Rouanet, Jean-Max

2007-09-19

273

Pollen tube NAD(P)H oxidases act as a speed control to dampen growth rate oscillations during polarized cell growth.  

PubMed

Reactive oxygen species (ROS) produced by NAD(P)H oxidases play a central role in plant stress responses and development. To better understand the function of NAD(P)H oxidases in plant development, we characterized the Arabidopsis thaliana NAD(P)H oxidases RBOHH and RBOHJ. Both proteins were specifically expressed in pollen and dynamically targeted to distinct and overlapping plasma membrane domains at the pollen tube tip. Functional loss of RBOHH and RBOHJ in homozygous double mutants resulted in reduced fertility. Analyses of pollen tube growth revealed remarkable differences in growth dynamics between Col-0 and rbohh-1 rbohj-2 pollen tubes. Growth rate oscillations of rbohh-1 rbohj-2 pollen tubes showed strong fluctuations in amplitude and frequency, ultimately leading to pollen tube collapse. Prior to disintegration, rbohh-1 rbohj-2 pollen tubes exhibit high-frequency growth oscillations, with significantly elevated growth rates, suggesting that an increase in the rate of cell-wall exocytosis precedes pollen tube collapse. Time-lapse imaging of exocytic dynamics revealed that NAD(P)H oxidases slow down pollen tube growth to coordinate the rate of cell expansion with the rate of exocytosis, thereby dampening the amplitude of intrinsic growth oscillations. Using the Ca(2+) reporter Yellow Cameleon 3.6, we demonstrate that high-amplitude growth rate oscillations in rbohh-1 rbohj-2 pollen tubes are correlated with growth-dependent Ca(2+) bursts. Electrophysiological experiments involving double mutant pollen tubes and pharmacological treatments also showed that ROS influence K(+) homeostasis. Our results indicate that, by limiting pollen tube growth, ROS produced by NAD(P)H oxidases modulate the amplitude and frequency of pollen tube growth rate oscillations. PMID:24506280

Lassig, Roman; Gutermuth, Timo; Bey, Till D; Konrad, Kai R; Romeis, Tina

2014-04-01

274

Increased accumulation of neutrophils and decreased fibrosis in the lung of NADPH oxidase-deficient C57BL/6 mice exposed to carbon nanotubes  

SciTech Connect

Single-walled carbon nanotubes (SWCNT) have been introduced into a large number of new technologies and consumer products. The combination of their exceptional features with very broad applications raised concerns regarding their potential health effects. The prime target for SWCNT toxicity is believed to be the lung where exposure may occur through inhalation, particularly in occupational settings. Our previous work has demonstrated that SWCNT cause robust inflammatory responses in rodents with very early termination of the acute phase and rapid onset of chronic fibrosis. Timely elimination of polymorphonuclear neutrophils (PMNs) through apoptosis and their subsequent clearance by macrophages is a necessary stage in the resolution of pulmonary inflammation whereby NADPH oxidase contributes to control of apoptotic cell death and clearance of PMNs. Thus, we hypothesized that NADPH oxidase may be an important regulator of the transition from the acute inflammation to the chronic fibrotic stage in response to SWCNT. To experimentally address the hypothesis, we employed NADPH oxidase-deficient mice which lack the gp91{sup phox} subunit of the enzymatic complex. We found that NADPH oxidase null mice responded to SWCNT exposure with a marked accumulation of PMNs and elevated levels of apoptotic cells in the lungs, production of pro-inflammatory cytokines, decreased production of the anti-inflammatory and pro-fibrotic cytokine, TGF-{beta}, and significantly lower levels of collagen deposition, as compared to C57BL/6 control mice. These results demonstrate a role for NADPH oxidase-derived reactive oxygen species in determining course of pulmonary response to SWCNT.

Shvedova, A.A. [Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, West Virginia University, Morgantown, WV (United States); Physiology and Pharmacology Department, West Virginia University, Morgantown, WV (United States)], E-mail: ats1@cdc.gov; Kisin, E.R. [Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, West Virginia University, Morgantown, WV (United States); Murray, A.R. [Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, West Virginia University, Morgantown, WV (United States); Physiology and Pharmacology Department, West Virginia University, Morgantown, WV (United States); Kommineni, C. [Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, West Virginia University, Morgantown, WV (United States); Castranova, V. [Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, West Virginia University, Morgantown, WV (United States); Physiology and Pharmacology Department, West Virginia University, Morgantown, WV (United States); Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA (United States); Fadeel, B. [Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institute Stockholm (Sweden); Kagan, V.E. [Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, West Virginia University, Morgantown, WV (United States); Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA (United States)

2008-09-01

275

Original article Variation for polyphenol oxidase activity  

E-print Network

Original article Variation for polyphenol oxidase activity in stems of Medicago species Michele) Abstract - Polyphenol oxidase (PPO) activity was detected in stems of both glandular-haired and glabrous from 120 to 180 kDa and the lighter one from 65 to 75 kDa. The substrate affinity for 4-methyl catechol

Paris-Sud XI, Université de

276

Hodgkin-Reed-Sternberg Cells in Classical Hodgkin Lymphoma Show Alterations of Genes Encoding the NADPH Oxidase Complex and Impaired Reactive Oxygen Species Synthesis Capacity  

PubMed Central

The membrane bound NADPH oxidase involved in the synthesis of reactive oxygen species (ROS) is a multi-protein enzyme encoded by CYBA, CYBB, NCF1, NCF2 and NCF4 genes. Growing evidence suggests a role of ROS in the modulation of signaling pathways of non-phagocytic cells, including differentiation and proliferation of B-cell progenitors. Transcriptional downregulation of the CYBB gene has been previously reported in cell lines of the B-cell derived classical Hodgkin lymphoma (cHL). Thus, we explored functional consequences of CYBB downregulation on the NADPH complex. Using flow cytometry to detect and quantify superoxide anion synthesis in cHL cell lines we identified recurrent loss of superoxide anion production in all stimulated cHL cell lines in contrast to stimulated non-Hodgkin lymphoma cell lines. As CYBB loss proved to exert a deleterious effect on the NADPH oxidase complex in cHL cell lines, we analyzed the CYBB locus in Hodgkin and Reed-Sternberg (HRS) cells of primary cHL biopsies by in situ hybridisation and identified recurrent deletions of the gene in 8/18 cases. Immunohistochemical analysis to 14 of these cases revealed a complete lack of detectable CYBB protein expression in all HRS cells in all cases studied. Moreover, by microarray profiling of cHL cell lines we identified additional alterations of NADPH oxidase genes including CYBA copy number loss in 3/7 cell lines and a significant downregulation of the NCF1 transcription (p=0.006) compared to normal B-cell subsets. Besides, NCF1 protein was significantly downregulated (p<0.005) in cHL compared to other lymphoma cell lines. Together this findings show recurrent alterations of the NADPH oxidase encoding genes that result in functional inactivation of the enzyme and reduced production of superoxide anion in cHL. PMID:24376854

Sosna, Justyna; Döring, Claudia; Klapper, Wolfram; Küppers, Ralf; Böttcher, Sebastian; Adam, Dieter; Siebert, Reiner; Schütze, Stefan

2013-01-01

277

NADPH oxidase complex and IBD candidate gene studies: identification of a rare variant in NCF2 that results in reduced binding to RAC2  

PubMed Central

Objective The NOX2 NADPH oxidase complex produces reactive oxygen species and plays a critical role in the killing of microbes by phagocytes. Genetic mutations in genes encoding components of the complex result in both X-linked and autosomal recessive forms of chronic granulomatous disease (CGD). Patients with CGD often develop intestinal inflammation that is histologically similar to Crohn's colitis, suggesting a common aetiology for both diseases. The aim of this study is to determine if polymorphisms in NOX2 NADPH oxidase complex genes that do not cause CGD are associated with the development of inflammatory bowel disease (IBD). Methods Direct sequencing and candidate gene approaches were used to identify susceptibility loci in NADPH oxidase complex genes. Functional studies were carried out on identified variants. Novel findings were replicated in independent cohorts. Results Sequence analysis identified a novel missense variant in the neutrophil cytosolic factor 2 (NCF2) gene that is associated with very early onset IBD (VEO-IBD) and subsequently found in 4% of patients with VEO-IBD compared with 0.2% of controls (p=1.3×10?5, OR 23.8 (95% CI 3.9 to 142.5); Fisher exact test). This variant reduced binding of the NCF2 gene product p67phox to RAC2. This study found a novel genetic association of RAC2 with Crohn's disease (CD) and replicated the previously reported association of NCF4 with ileal CD. Conclusion These studies suggest that the rare novel p67phox variant results in partial inhibition of oxidase function and are associated with CD in a subgroup of patients with VEO-IBD; and suggest that components of the NADPH oxidase complex are associated with CD. PMID:21900546

Muise, Aleixo M; Xu, Wei; Guo, Cong-Hui; Walters, Thomas D; Wolters, Victorien M; Fattouh, Ramzi; Lam, Grace Y; Hu, Pingzhao; Murchie, Ryan; Sherlock, Mary; Gana, Juan Cristobal; Russell, Richard K; Glogauer, Michael; Duerr, Richard H; Cho, Judy H; Lees, Charlie W; Satsangi, Jack; Wilson, David C; Paterson, Andrew D; Griffiths, Anne M; Silverberg, Mark S; Brumell, John H

2013-01-01

278

Isoproterenol disperses distribution of NADPH oxidase, MMP-9, and pPKC? in the heart, which are mitigated by endothelin receptor antagonist CPU0213  

PubMed Central

Aim: Spatial dispersion of bioactive substances in the myocardium could serve as pathological basis for arrhythmogenesis and cardiac impairment by ?-adrenoceptor stimulation. We hypothesized that dispersed NADPH oxidase, protein kinase C? (PKC?), early response gene (ERG), and matrix metalloproteinase 9(MMP-9) across the heart by isoproterenol (ISO) medication might be mediated by the endothelin (ET) – ROS pathway. We aimed to verify if ISO induced spatially heterogeneous distribution of pPKC?, NAPDH oxidase, MMP-9 and ERG could be mitigated by either an ET receptor antagonist CPU0213 or iNOS inhibitor aminoguanidine. Methods: Rats were treated with ISO (1 mg/kg sc) for 10 days, and drug interventions (mg/kg) either CPU0213 (30 sc) or aminoguanidine (100 ip) were administered on days 8–10. Expression of NADPH oxidase, MMP-9, ERG, and PKC? in the left and right ventricle (LV, RV) and septum (S) were measured separately. Results: Ventricular hypertrophy was found in the LV, S, and RV, in association with dispersed QTc and oxidative stress in ISO-treated rats. mRNA and protein expression of MMP-9, PKC?, NADPH oxidase and ERG in the LV, S, and RV were obviously dispersed, with augmented expression mainly in the LV and S. Dispersed parameters were re-harmonized by either CPU0213, or aminoguanidine. Conclusion: We found at the first time that ISO-induced dispersed distribution of pPKC?, NADPH oxidase, MMP-9, and ERG in the LV, S, and RV of the heart, which were suppressed by either CPU0213 or aminoguanidine. It indicates that the ET-ROS pathway plays a role in the dispersed distribution of bioactive substances following sustained ?-receptor stimulation. PMID:19597524

Cheng, Yu-si; Dai, De-zai; Dai, Yin

2009-01-01

279

The NADPH oxidase NOX2 plays a role in periodontal pathologies  

Microsoft Academic Search

Oxidative stress plays an important role in periodontal health and disease. The phagocyte nicotinamide adenine dinucleotide\\u000a phosphate oxidase NOX2 is most likely one of the key sources of reactive oxygen species (ROS) in periodontal tissues. This\\u000a review will discuss three clinical aspects of NOX2 function. We will first focus on oral pathology in NOX2 deficiency such\\u000a as chronic granulomatous disease

Catherine Giannopoulou; Karl-Heinz Krause; Frauke Müller

2008-01-01

280

BRAINA JOURNAL OF NEUROLOGY NADPH oxidase expression in active multiple  

E-print Network

Horssen2, * and Hans Lassmann1, * 1 Centre for Brain Research, Medical University of Vienna, A-1090 Wien authors contributed equally to this work Correspondence to: Prof. Dr Hans Lassmann, Centre for Brain Research, Medical University of Vienna, Spitalgasse 4, A-1090 Wien, Austria E-mail: hans.lassmann

Hayar, Abdallah

281

The C242T-polymorphism of the NADPH\\/NADH oxidase gene p22phox subunit is not associated with pre-eclampsia  

Microsoft Academic Search

Pre-eclampsia is a pregnancy-related multisystem disorder characterised by elevation of blood pressure and proteinuria, in which oxidative stress may play an important role. Blood pressure is partly controlled by O?2 production by NADPH\\/NADH oxidase and recently it was shown that a C242T substitution in the p22phox gene was associated with coronary artery disease, in which elevated blood pressure and oxidative

M T M Raijmakers; E M Roes; E A P Steegers; W H M Peters; WHM Peters

2002-01-01

282

Effects of the NADPH oxidase p22phox C242T polymorphism on endurance exercise performance and oxidative DNA damage in response to aerobic exercise training  

Microsoft Academic Search

We examined the effects of the NADPH oxidase p22phox C242T polymorphism on endurance exercise performance and oxidative DNA\\u000a damage in response to acute and chronic exercises. One hundred three subjects were recruited, among which 26 healthy subjects\\u000a (CC: 12, TC: 12, and TT: 2) were studied during rest, exercise at 85% VO2max, and recovery before and after 8 weeks of

Il-Young Paik; Chan-Ho Jin; Hwa-Eun Jin; Young-Il Kim; Su-Youn Cho; Hee-Tae Roh; Ah-Ram Suh; Sang-Hoon Suh

2009-01-01

283

Arterial flow reduces oxidative stress via an antioxidant response element and Oct-1 binding site within the NADPH oxidase 4 promoter in endothelial cells  

Microsoft Academic Search

The main sources of oxidative stress in the vessel wall are nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox)\\u000a complexes. The endothelium mainly expresses the Nox4-containing complex; however, the mechanism by which shear stress in endothelial\\u000a cells regulates Nox4 is not well understood. This study demonstrates that long-term application of arterial laminar shear\\u000a stress using a cone-and-plate viscometer reduces endothelial superoxide

Claudia GoettschWinfried; Winfried Goettsch; Melanie Brux; Claudia Haschke; Coy Brunssen; Gregor Muller; Stefan R. Bornstein; Nicole Duerrschmidt; Andreas H. Wagner; Henning Morawietz

2011-01-01

284

Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent ?1-subunit glutathionylation: implications for angiotensin II-induced vascular dysfunction.  

PubMed

Glutathionylation of the Na(+)-K(+) pump's ?1-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na(+)-K(+) pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific ?-subunit isoform expression and receptor-coupled pathways. As Na(+)-K(+) pump activity is an important determinant of vascular tone through effects on [Ca(2+)]i, we have examined the role of oxidative regulation of the Na(+)-K(+) pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. ?1-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na(+)-K(+)ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K(+)-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na(+)-K(+) ATPase and decrease in K(+)-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of ?1-subunit. Knock-out of FXYD1 dramatically decreased K(+)-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased ?1-subunit glutathionylation, and enhanced K(+)-induced vasorelaxation. Ang II inhibits the Na(+)-K(+) pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump's ?1-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na(+)-K(+) pump and may have an important protective role in the vasculature under conditions of oxidative stress. PMID:23816524

Liu, Chia-Chi; Karimi Galougahi, Keyvan; Weisbrod, Robert M; Hansen, Thomas; Ravaie, Ramtin; Nunez, Andrea; Liu, Yi B; Fry, Natasha; Garcia, Alvaro; Hamilton, Elisha J; Sweadner, Kathleen J; Cohen, Richard A; Figtree, Gemma A

2013-12-01

285

NADPH Oxidase-2 Derived ROS Dictates Murine DC Cytokine-Mediated Cell Fate Decisions during CD4 T Helper-Cell Commitment  

PubMed Central

NADPH oxidase-2 (Nox2)/gp91phox and p47phox deficient mice are prone to hyper-inflammatory responses suggesting a paradoxical role for Nox2-derived reactive oxygen species (ROS) as anti-inflammatory mediators. The molecular basis for this mode of control remains unclear. Here we demonstrate that IFN?/LPS matured p47phox?/?-ROS deficient mouse dendritic cells (DC) secrete more IL-12p70 than similarly treated wild type DC, and in an in vitro co-culture model IFN?/LPS matured p47phox?/? DC bias more ovalbumin-specific CD4+ T lymphocytes toward a Th1 phenotype than wild type (WT) DC through a ROS-dependent mechanism linking IL-12p70 expression to regulation of p38-MAPK activation. The Nox2-dependent ROS production in DC negatively regulates proinflammatory IL-12 expression in DC by constraining p38-MAPK activity. Increasing endogenous H2O2 attenuates p38-MAPK activity in IFN?/LPS stimulated WT and p47phox?/? DC, which suggests that endogenous Nox 2-derived ROS functions as a secondary messenger in the activated p38-MAPK signaling pathway during IL-12 expression. These findings indicate that ROS, generated endogenously by innate and adaptive immune cells, can function as important secondary messengers that can regulate cytokine production and immune cell cross-talk to control during the inflammatory response. PMID:22145029

Wood, Adam; Zhu, Nannan; Zhao, Yongge; Koontz, Sherry M.; Jackson, Sharon H.

2011-01-01

286

Diapocynin, a Dimer of the NADPH Oxidase Inhibitor Apocynin, Reduces ROS Production and Prevents Force Loss in Eccentrically Contracting Dystrophic Muscle  

PubMed Central

Elevation of intracellular Ca2+, excessive ROS production and increased phospholipase A2 activity contribute to the pathology in dystrophin-deficient muscle. Moreover, Ca2+, ROS and phospholipase A2, in particular iPLA2, are thought to potentiate each other in positive feedback loops. NADPH oxidases (NOX) have been considered as a major source of ROS in muscle and have been reported to be overexpressed in muscles of mdx mice. We report here on our investigations regarding the effect of diapocynin, a dimer of the commonly used NOX inhibitor apocynin, on the activity of iPLA2, Ca2+ handling and ROS generation in dystrophic myotubes. We also examined the effects of diapocynin on force production and recovery ability of isolated EDL muscles exposed to eccentric contractions in vitro, a damaging procedure to which dystrophic muscle is extremely sensitive. In dystrophic myotubes, diapocynin inhibited ROS production, abolished iPLA2 activity and reduced Ca2+ influx through stretch-activated and store-operated channels, two major pathways responsible for excessive Ca2+ entry in dystrophic muscle. Diapocynin also prevented force loss induced by eccentric contractions of mdx muscle close to the value of wild-type muscle and reduced membrane damage as seen by Procion orange dye uptake. These findings support the central role played by NOX-ROS in the pathogenic cascade leading to muscular dystrophy and suggest diapocynin as an effective NOX inhibitor that might be helpful for future therapeutic approaches. PMID:25329652

Ismail, Hesham M.; Scapozza, Leonardo; Ruegg, Urs T.; Dorchies, Olivier M.

2014-01-01

287

Dual Roles of Reactive Oxygen Species and NADPH Oxidase RBOHD in an Arabidopsis-Alternaria Pathosystem1[W  

PubMed Central

Arabidopsis (Arabidopsis thaliana) NADPH oxidases have been reported to suppress the spread of pathogen- and salicylic acid-induced cell death. Here, we present dual roles of RBOHD (for respiratory burst oxidase homolog D) in an Arabidopsis-Alternaria pathosystem, suggesting either initiation or prevention of cell death dependent on the distance from pathogen attack. Our data demonstrate that a rbohD knockout mutant exhibits increased spread of cell death at the macroscopic level upon inoculation with the fungus Alternaria brassicicola. However, the cellular patterns of reactive oxygen species accumulation and cell death are fundamentally different in the AtrbohD mutant compared with the wild type. Functional RBOHD causes marked extracellular hydrogen peroxide accumulation as well as cell death in distinct, single cells of A. brassicicola-infected wild-type plants. This single cell response is missing in the AtrbohD mutant, where infection triggers spreading-type necrosis preceded by less distinct chloroplastic hydrogen peroxide accumulation in large clusters of cells. While the salicylic acid analog benzothiadiazole induces the action of RBOHD and the development of cell death in infected tissues, the ethylene inhibitor aminoethoxyvinylglycine inhibits cell death, indicating that both salicylic acid and ethylene positively regulate RBOHD and cell death. Moreover, A. brassicicola-infected AtrbohD plants hyperaccumulate ethylene and free salicylic acid compared with the wild type, suggesting negative feedback regulation of salicylic acid and ethylene by RBOHD. We propose that functional RBOHD triggers death in cells that are damaged by fungal infection but simultaneously inhibits death in neighboring cells through the suppression of free salicylic acid and ethylene levels. PMID:19726575

Pogany, Miklos; von Rad, Uta; Grun, Sebastian; Dongo, Anita; Pintye, Alexandra; Simoneau, Philippe; Bahnweg, Gunther; Kiss, Levente; Barna, Balazs; Durner, Jorg

2009-01-01

288

Chlorella Induces Stomatal Closure via NADPH Oxidase-Dependent ROS Production and Its Effects on Instantaneous Water Use Efficiency in Vicia faba  

PubMed Central

Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels. PMID:24687099

Li, Yan; Xu, Shan-Shan; Gao, Jing; Pan, Sha; Wang, Gen-Xuan

2014-01-01

289

Role of NADPH oxidase isoforms NOX1, NOX2 and NOX4 in myocardial ischemia/reperfusion injury.  

PubMed

Myocardial reperfusion injury is mediated by several processes including increase of reactive oxygen species (ROS). The aim of the study is to identify potential sources of ROS contributing to myocardial ischemia-reperfusion injury. For this purpose, we investigated myocardial ischemia/reperfusion pathology in mice deficient in various NADPH oxidase isoforms (Nox1, Nox2, Nox4, as well as Nox1/2 double knockout). Following 30min of ischemia and 24h of reperfusion, a significant decrease in the size of myocardial infarct was observed in Nox1-, Nox2- and Nox1/Nox2-, but not in Nox4-deficient mice. However, no protection was observed in a model of chronic ischemia, suggesting that NOX1 and NOX2-mediated oxidative damage occurs during reperfusion. Cardioprotective effect of Nox1 and Nox2 deficiencies was associated with decrease of neutrophil invasion, but, on the other hand an improved reperfusion injury was also observed in isolated perfused hearts (Langendorff model) suggesting that inflammatory cells were not the major source of oxidative damage. A decrease in global post-reperfusion oxidative stress was clearly detected in Nox2-, but not in Nox1-deficient hearts. Analysis of key signaling pathways during reperfusion suggests distinct cardioprotective patterns: increased phosphorylation was seen for Akt and Erk in Nox1-deficient mice and for Stat3 and Erk in Nox2-deficient mice. Consequently, NOX1 and NOX2 represent interesting drug targets for controlling reperfusion damage associated with revascularization in coronary disease. PMID:24051369

Braunersreuther, Vincent; Montecucco, Fabrizio; Asrih, Mohamed; Ashri, Mohammed; Pelli, Graziano; Galan, Katia; Frias, Miguel; Burger, Fabienne; Quinderé, Ana Luíza Gomez; Montessuit, Christophe; Krause, Karl-Heinz; Mach, François; Jaquet, Vincent

2013-11-01

290

NADPH oxidase-dependent oxidative stress and mitochondrial damage in hippocampus of D-galactose-induced aging rats.  

PubMed

Mitochondrial DNA (mtDNA) common deletion (CD) plays a significant role in aging and age-related diseases. In this study, we used D-galactose (D-gal) to generate an animal model of aging and the involvement and causative mechanisms of mitochondrial damage in such a model were investigated. Twenty 5-week-old male Sprague-Dawley rats were randomly divided into two groups: D-gal group (n=10) and control group (n=10). The quantity of the mtDNA CD in the hippocampus was determined using a TaqMan real-time PCR assay. Transmission electron microscopy was used to observe the mitochondrial ultrastructure in the hippocampus. Western blot was used to detect the protein levels of NADPH oxidase (NOX) and uncoupling protein 2 (UCP2). We found that the level of mtDNA CD was significantly higher in the hippocampus of D-gal-induced aging rats than in control rats. In comparison with the control group, the mitochondrial ultrastructure in the hippocampus of D-gal-treated rats was damaged, and the protein levels of NOX and UCP2 were significantly increased in the hippocampus of D-gal-induced aging rats. This study demonstrated that the levels of mtDNA CD and NOX protein expression were significantly increased in the hippocampus of D-gal-induced aging rats. These findings indicate that NOX-dependent reactive oxygen species generation may contribute to D-gal-induced mitochondrial damage. PMID:22886955

Du, Zhengde; Hu, Yujuan; Yang, Yang; Sun, Yu; Zhang, Sulin; Zhou, Tao; Zeng, Lingling; Zhang, Wenjuan; Huang, Xiang; Kong, Weijia; Zhang, Honglian

2012-08-01

291

NADPH phagocyte oxidase knockout mice control Trypanosoma cruzi proliferation, but develop circulatory collapse and succumb to infection.  

PubMed

(•)NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91(phox) (-/-) or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-? and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with (•)NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi. PMID:22348160

Santiago, Helton C; Gonzalez Lombana, Claudia Z; Macedo, Juan P; Utsch, Lara; Tafuri, Wagner L; Campagnole-Santos, Maria José; Alves, Rosana O; Alves-Filho, José C F; Romanha, Alvaro J; Cunha, Fernando Queiroz; Teixeira, Mauro M; Radi, Rafael; Vieira, Leda Q

2012-01-01

292

A point mutation in gp91-phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox.  

PubMed Central

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patient's neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp91-phox inhibited NADPH oxidase activity in the cell-free assay (IC50 about 10 microM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox. Images PMID:8182143

Leusen, J H; de Boer, M; Bolscher, B G; Hilarius, P M; Weening, R S; Ochs, H D; Roos, D; Verhoeven, A J

1994-01-01

293

Antioxidant protection by PECAM-targeted delivery of a novel NADPH-oxidase inhibitor to the endothelium in vitro and in vivo.  

PubMed

Oxidant stress caused by pathological elevation of reactive oxygen species (ROS) production in the endothelial cells lining the vascular lumen is an important component of many vascular and pulmonary disease conditions. NADPH oxidase (NOX) activated by pathological mediators including angiotensin and cytokines is a major source of endothelial ROS. In order to intercept this pathological pathway, we have encapsulated an indirect NOX inhibitor, MJ33, into immunoliposomes (Ab-MJ33/IL) targeted to endothelial marker platelet endothelial cell adhesion molecule (PECAM-1). Ab-MJ33/IL, but not control IgG-MJ33/IL are specifically bound to endothelium and attenuated angiotensin-induced ROS production in vitro and in vivo. Additionally, Ab-MJ33/IL inhibited endothelial expression of the inflammatory marker vascular cell adhesion molecule (VCAM) in cells and animals challenged with the cytokine TNF. Furthermore, Ab-MJ33/IL alleviated pathological disruption of endothelial permeability barrier function in cells exposed to vascular endothelial growth factor (VEGF) and in the lungs of mice challenged with lipopolysaccharide (LPS). Of note, the latter beneficial effect has been achieved both by prophylactic and therapeutic injection of Ab-MJ33/IL in animals. Therefore, specific suppression of ROS production by NOX in endothelium, attainable by Ab-MJ33/IL targeting, may help deciphering mechanisms of vascular oxidative stress and inflammation, and potentially improve treatment of these conditions. PMID:22974832

Hood, Elizabeth D; Greineder, Colin F; Dodia, Chandra; Han, Jingyan; Mesaros, Clementina; Shuvaev, Vladimir V; Blair, Ian A; Fisher, Aron B; Muzykantov, Vladimir R

2012-10-28

294

Sulfite oxidase activity in Thiobacillus novellus.  

PubMed

Thiobacillus novellus shows a maximum induction of sulfite oxidase activity and a maximum growth rate as a result of supplementing the autotrophic growth medium with 4.0 microM ammonium molybdate. Cells grown in the presence of molybdate showed approximately 10-fold increases in the amount of enzyme-associated molybdenum and in the sulfite-to-cytochrome c and sulfite-to-ferricyanide reductase activities. The effect of exogenous molybdate was not discernible with cells grown in the absence of thiosulfate. Tungsten inhibited the growth of T. novellus and the expression of sulfite oxidase activity. PMID:6630156

Southerland, W M; Toghrol, F

1983-11-01

295

The NADPH Oxidase Complexes in Botrytis cinerea: Evidence for a Close Association with the ER and the Tetraspanin Pls1  

PubMed Central

NADPH oxidases (Nox) are major enzymatic systems that generate reactive-oxygen species (ROS) in multicellular eukaryotes. In several fungi they have been shown to be involved in sexual differentiation and pathogenicity. However, in contrast to the well characterized mammalian systems, basic information on the composition, recruitment, and localization of fungal Nox complexes and on the molecular mechanisms of their cellular effects are still lacking. Here we give a detailed analysis of components of the Nox complexes in the gray mold fungus Botrytis cinerea. It had previously been shown that the two catalytic transmembrane subunits BcNoxA and B are important for development of sclerotia and for full virulence, with BcNoxA being involved in spreading of lesions and BcNoxB in penetration; BcNoxR functions as a regulator of both subunits. Here we present evidence (using for the first time a functional GFP fusion able to complement the ?bcnoxA mutant) that BcNoxA localizes mainly to the ER and at the plasma membrane; BcNoxB shows a similar localization pattern, while the regulator BcNoxR is found in vesicles throughout the hyphae and at the hyphal tip. To identify possible interaction partners, which could be involved in the localization or recruitment of the Nox complexes, we functionally characterized the tetraspanin Pls1, a transmembrane protein, which had been suggested to be a NoxB-interacting partner in the saprophyte Podospora anserina. Knock-out experiments and GFP fusions substantiate a link between BcNoxB and BcPls1 because both deletion mutants have overlapping phenotypes (especially a defect in penetration), and the proteins show a similar localization pattern (ER). However, in contrast to the corresponding protein in P. anserina BcPls1 is important for female fertility, but not for ascospore germination. PMID:23418468

Siegmund, Ulrike; Heller, Jens; van Kann, Jan A. L.; Tudzynski, Paul

2013-01-01

296

156Pro-->Gln substitution in the light chain of cytochrome b558 of the human NADPH oxidase (p22-phox) leads to defective translocation of the cytosolic proteins p47-phox and p67-phox  

PubMed Central

Src homology 3 (SH3) domains have been suggested to play an important role in the assembly of the superoxide-forming nicotinamide adenine dinucleotide phosphate (NADPH) oxidase upon activation of phagocytes, which involves the association of membrane-bound and cytosolic components. We studied the translocation of the cytosolic proteins to the plasma membrane in neutrophils of a patient with a point mutation in the gene encoding the light chain of cytochrome b558. This mutation leads to a substitution at residue 156 of a proline into a glutamine in a putative SH3 binding domain of p22-phox (Dinauer, M., E. A. Pierce, R. W. Erickson, T. Muhlebach, H. Messner, R. A. Seger, S. H. Orkin, and J. T. Curnutte. 1991. Proc. Natl. Acad. Sci. 88:11231). In PMA- stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient's neutrophils was virtually absent. In contrast, when solubilized membranes of the patient's neutrophils were activated with phospholipids in the absence of cytosol (Koshkin, V., and E. Pick. 1993. FEBS [Fed. Eur. Biochem. Soc.] Lett. 327:57), the rate of NADPH- dependent oxygen uptake was observed at a rate similar to that of control membranes. We suggest that the binding of an SH3 domain of p47- phox to p22-phox, and thus activation of the oxidase, does not occur in the neutrophils of this patient, although under artificial conditions, electron flow from NADPH to oxygen in cytochrome b558 is possible. PMID:7964505

1994-01-01

297

Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion  

NASA Technical Reports Server (NTRS)

Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of O2- from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47phox-/- (MAE-p47-/-) mice, which lack a component of NAD(P)H oxidase. O2- production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (+/- 5 dynes/cm2) increased O2- (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm2, 18 h) significantly decreased both monocyte adhesion and O2- compared with static conditions. In contrast, neither LS nor OS were able to induce O2- production and monocyte adhesion to MAE-p47-/-. Treating MAE-C57 with a cell-permeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47phox in MAE-p47-/- restored OS-induced O2- production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates O2- and/or its derivatives produced from p47phox-dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.

Hwang, Jinah; Saha, Aniket; Boo, Yong Chool; Sorescu, George P.; McNally, J. Scott; Holland, Steven M.; Dikalov, Sergei; Giddens, Don P.; Griendling, Kathy K.; Harrison, David G.; Jo, Hanjoong

2003-01-01

298

Ethers and esters derived from apocynin avoid the interaction between p47phox and p22phox subunits of NADPH oxidase: evaluation in vitro and in silico  

PubMed Central

NOX (NADPH oxidase) plays an important role during several pathologies because it produces the superoxide anion (O2•?), which reacts with NO (nitric oxide), diminishing its vasodilator effect. Although different isoforms of NOX are expressed in ECs (endothelial cells) of blood vessels, the NOX2 isoform has been considered the principal therapeutic target for vascular diseases because it can be up-regulated by inhibiting the interaction between its p47phox (cytosolic protein) and p22phox (transmembrane protein) subunits. In this research, two ethers, 4-(4-acetyl-2-methoxy-phenoxy)-acetic acid (1) and 4-(4-acetyl-2-methoxy-phenoxy)-butyric acid (2) and two esters, pentanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester (3) and heptanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester (4), which are apocynin derivatives were designed, synthesized and evaluated as NOX inhibitors by quantifying O2•? production using EPR (electron paramagnetic resonance) measurements. In addition, the antioxidant activity of apocynin and its derivatives were determined. A docking study was used to identify the interactions between the NOX2?s p47phox subunit and apocynin or its derivatives. The results showed that all of the compounds exhibit inhibitory activity on NOX, being 4 the best derivative. However, neither apocynin nor its derivatives were free radical scavengers. On the other hand, the in silico studies demonstrated that the apocynin and its derivatives were recognized by the polybasic SH3A and SH3B domains, which are regions of p47phox that interact with p22phox. Therefore this experimental and theoretical study suggests that compound 4 could prevent the formation of the complex between p47phox and p22phox without needing to be activated by MPO (myeloperoxidase), this being an advantage over apocynin. PMID:23802190

Macias-Perez, Martha Edith; Martinez-Ramos, Federico; Padilla-Martinez, Itzia Irene; Correa-Basurto, Jose; Kispert, Lowell; Mendieta-Wejebe, Jessica Elena; Rosales-Hernandez, Martha Cecilia

2013-01-01

299

Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen  

Microsoft Academic Search

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O3) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O3 fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide

Stefania Pasqualini; Emma Tedeschini; Giuseppe Frenguelli; Nicole Wopfner; Fatima Ferreira; Gennaro D’Amato; Luisa Ederli

2011-01-01

300

Targeting Vascular NADPH Oxidase 1 Blocks Tumor Angiogenesis through a PPAR? Mediated Mechanism  

PubMed Central

Reactive oxygen species, ROS, are regulators of endothelial cell migration, proliferation and survival, events critically involved in angiogenesis. Different isoforms of ROS-generating NOX enzymes are expressed in the vasculature and provide distinct signaling cues through differential localization and activation. We show that mice deficient in NOX1, but not NOX2 or NOX4, have impaired angiogenesis. NOX1 expression and activity is increased in primary mouse and human endothelial cells upon angiogenic stimulation. NOX1 silencing decreases endothelial cell migration and tube-like structure formation, through the inhibition of PPAR?, a regulator of NF-?B. Administration of a novel NOX-specific inhibitor reduced angiogenesis and tumor growth in vivo in a PPAR? dependent manner. In conclusion, vascular NOX1 is a critical mediator of angiogenesis and an attractive target for anti-angiogenic therapies. PMID:21326871

Garrido-Urbani, Sarah; Jemelin, Stephane; Deffert, Christine; Carnesecchi, Stephanie; Basset, Olivier; Szyndralewiez, Cedric; Heitz, Freddy; Page, Patrick; Montet, Xavier; Michalik, Liliane; Arbiser, Jack; Ruegg, Curzio; Krause, Karl Heinz; Imhof, Beat

2011-01-01

301

Hyperosmolarity-induced up-regulation of claudin-4 mediated by NADPH oxidase-dependent H2O2 production and Sp1/c-Jun cooperation.  

PubMed

Claudin-4 is exclusively localized in the tight collecting ducts in the renal tubule. We examined what molecular mechanism is involved in the regulation of claudin-4 expression. In Madin-Darby canine kidney cells, hyperosmolarity increased the expression level of claudin-4 and the production of reactive oxygen species, which were inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, and manganese (III) tetrakis (4-benzoic acid)porphyrin (MnTBAP), a scavenger of H2O2. Both hyperosmolarity and H2O2 increased p-ERK1/2 and p-JNK, which were inhibited by U0126, a MEK inhibitor, and SP600125, a JNK inhibitor, respectively. Immunoprecipitation assay showed that hyperosmolarity increased the association of nuclear Sp1 with c-Jun, which was inhibited by U0126 and SP600125. In mouse inner medullary collecting duct cells and rat kidney slices, hyperosmolarity increased the expression level of claudin-4, which was inhibited by DPI, MnTBAP, U0126, and SP600125. Hyperosmolarity increased luciferase reporter activity of claudin-4, which was inhibited by U0126, SP600125, Sp1 siRNA, and c-Jun siRNA. The activity was inhibited by the mutation in the Sp1 binding site. Chromatin immunoprecipitation assay and avidin-biotin conjugated DNA assay showed that Sp1 and c-Jun are associated with the Sp1 binding site. These results suggest that hyperosmolarity increases nuclear Sp1/c-Jun complex and the association of the complex with the Sp1 binding site, resulting in the segment-specific expression of claudin-4 in the kidney. PMID:23816505

Ikari, Akira; Atomi, Kosuke; Yamazaki, Yasuhiro; Sakai, Hideki; Hayashi, Hisayoshi; Yamaguchi, Masahiko; Sugatani, Junko

2013-12-01

302

XANTHINE OXIDASE ACTIVITY IN REGENERATING LIVER*  

E-print Network

The xanthine oxiclase activity of mouse regenerating liver has been shown to be elevated during the period of rapid liver growth and proliferation. This increase is evident when the enzyme activity is expressed per unit wet tissue weight, per unit nitrogen, or per cell. The adrenal cortex probably plays only a minor role in implementing this phenomenon. Further augmentation of the xanthine oxidase level of regenerating liver is not induced by the administration of large quantities of the substrate, xanthine, to the animal.

Muriel Feigelson; Philip Feigelson; Paul; R. Gross; Ab Stract

303

Angiotensin II modulates mouse skeletal muscle resting conductance to chloride and potassium ions and calcium homeostasis via the AT1 receptor and NADPH oxidase.  

PubMed

Angiotensin II (ANG II) plays a role in muscle wasting and remodeling; however, little evidence shows its direct effects on specific muscle functions. We presently investigated the acute in vitro effects of ANG II on resting ionic conductance and calcium homeostasis of mouse extensor digitorum longus (EDL) muscle fibers, based on previous findings that in vivo inhibition of ANG II counteracts the impairment of macroscopic ClC-1 chloride channel conductance (gCl) in the mdx mouse model of muscular dystrophy. By means of intracellular microelectrode recordings we found that ANG II reduced gCl in the nanomolar range and in a concentration-dependent manner (EC50 = 0.06 ?M) meanwhile increasing potassium conductance (gK). Both effects were inhibited by the ANG II receptors type 1 (AT1)-receptor antagonist losartan and the protein kinase C inhibitor chelerythrine; no antagonism was observed with the AT2 antagonist PD123,319. The scavenger of reactive oxygen species (ROS) N-acetyl cysteine and the NADPH-oxidase (NOX) inhibitor apocynin also antagonized ANG II effects on resting ionic conductances; the ANG II-dependent gK increase was blocked by iberiotoxin, an inhibitor of calcium-activated potassium channels. ANG II also lowered the threshold for myofiber and muscle contraction. Both ANG II and the AT1 agonist L162,313 increased the intracellular calcium transients, measured by fura-2, with a two-step pattern. These latter effects were not observed in the presence of losartan and of the phospholipase C inhibitor U73122 and the in absence of extracellular calcium, disclosing a Gq-mediated calcium entry mechanism. The data show for the first time that the AT1-mediated ANG II pathway, also involving NOX and ROS, directly modulates ion channels and calcium homeostasis in adult myofibers. PMID:25080489

Cozzoli, Anna; Liantonio, Antonella; Conte, Elena; Cannone, Maria; Massari, Ada Maria; Giustino, Arcangela; Scaramuzzi, Antonia; Pierno, Sabata; Mantuano, Paola; Capogrosso, Roberta Francesca; Camerino, Giulia Maria; De Luca, Annamaria

2014-10-01

304

Long term liver specific glucokinase gene defect induced diabetic cardiomyopathy by up regulating NADPH oxidase and down regulating insulin receptor and p-AMPK  

PubMed Central

Background The liver-specific glucokinase knockout (gckw/–) mouse experiences long-term hyperglycemia and insulin resistance. This study was designed to evaluate the functional and structural changes in the myocardium of 60 week-old gckw/– mice, and to investigate the effect of rosiglitazone on the myocardium in this model. Methods 60 week-old gckw/– mice were randomly divided into 3 groups: gckw/–, gckw/– mice treated with insulin (1 U/kg) and gckw/– mice treated with rosiglitazone (18 mg/kg). Insulin or rosiglitazone treatment was for 4 weeks. Gckw/w litermates were used as controls. Echocardiography, electrocardiogram, biochemical, histopathological, ultrastructural, real time PCR and Western blot studies were performed to examine for structural and functional changes. Results Long-term liver-specific gck knockout in mice elicits hyperglycaemia and insulin resistance. Compared to age matched gckw/w mice, 60 week-old gckw/– mice showed decreased LV internal dimension, increased posterior wall thickness, lengthened PR and QRS intervals, up-regulated MLC2 protein expression, decreased SOD activity, increased MDA levels and up-regulated Cyba mRNA. Morphological studies revealed that there was an increase in the amount of PAS and Masson positively stained material, as did the number and proportion of the cell occupied by mitochondria in the gckw/– mice. Western blot analysis revealed that the levels of the insulin receptor, Akt, phosphorylated AMPK beta and phosphorylated ACC were reduced in gckw/– mice. These effects were partly attenuated or ablated by treatment with rosiglitazone. Conclusions Our results indicate that changes in the myocardium occur in the liver-specific glucokinase knockout mouse and suggest that reduced glucokinase expression in the liver may induce diabetic cardiomyopathy by up regulating NADPH oxidase and down regulating insulin receptor and p-AMPK protein levels. Rosiglitazone treatment may protect against diabetic cardiomyopathy by altering the levels of a set of proteins involved in cardiac damage. PMID:24447392

2014-01-01

305

A commercial extract of fruits and vegetables, Oxxynea, acts as a powerful antiatherosclerotic supplement in an animal model by reducing cholesterolemia, oxidative stress, and NADPH oxidase expression.  

PubMed

The effects of fruit and vegetable extract (Oxxynea) on plasma cholesterol, early atherosclerosis, cardiac production of superoxide anion, and NAD(P)H oxidase expression were studied in an animal model of atherosclerosis. Thirty six hamsters were divided into two groups of 18 and fed an atherogenic diet for 12 weeks. They received by gavage either water or Oxxynea in water at a human dose equivalent of 10 fruits and vegetables per day. Oxxynea lowered plasma cholesterol and non-HDL cholesterol, but not HDL-cholesterol, and increased plasma antioxidant capacity. It also strongly reduced the area of aortic fatty streak deposition by 77%, cardiac production of superoxide anion by 45%, and p22phox subunit of NAD(P)H oxidase expression by 59%. These findings support the view that chronic consumption of antioxidants supplied by fruits and vegetables has potential beneficial effects with respect to the development of atherosclerosis. The underlying mechanism is related mainly to inhibiting pro-oxidant factors and improving the serum lipid profile. PMID:17444653

Sutra, Thibault; Décordé, Kelly; Riss, Jérome; Dallas, Constant; Cristol, Jean-Paul; Rouanet, Jean-Max

2007-05-16

306

Purification of active recombinant trypanosome alternative oxidase  

Microsoft Academic Search

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain in long slender bloodstream forms of African trypanosomes. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase. These characteristics are distinct from those of the bacterial quinol oxidases, proteins that belong to the heme-copper terminal oxidase superfamily. The inability to purify stable TAO has severely hampered biochemical studies of the

Coichi Nihei; Yoshihisa Fukai; Keisuke Kawai; Arihiro Osanai; Yoshisada Yabu; Takashi Suzuki; Nobuo Ohta; Nobuko Minagawa; Kazuo Nagai; Kiyoshi Kita

2003-01-01

307

Effects of N-Acetylcysteine on Nicotinamide Dinucleotide Phosphate Oxidase Activation and Antioxidant Status in Heart, Lung, Liver and Kidney in Streptozotocin-Induced Diabetic Rats  

PubMed Central

Purpose Hyperglycemia increases reactive oxygen species (ROS) and the resulting oxidative stress plays a key role in the pathogenesis of diabetic complications. Nicotinamide dinucleotide phosphate (NADPH) oxidase is one of the major sources of ROS production in diabetes. We, therefore, examined the possibility that NADPH oxidase activation is increased in various tissues, and that the antioxidant N-acetylcysteine (NAC) may have tissue specific effects on NADPH oxidase and tissue antioxidant status in diabetes. Materials and Methods Control (C) and streptozotocin-induced diabetic (D) rats were treated either with NAC (1.5 g/kg/day) orally or placebo for 4 weeks. The plasma, heart, lung, liver, kidney were harvested immediately and stored for biochemical or immunoblot analysis. Results levels of free 15-F2t-isoprostane were increased in plasma, heart, lung, liver and kidney tissues in diabetic rats, accompanied with significantly increased membrane translocation of the NADPH oxidase subunit p67phox in all tissues and increased expression of the membrane-bound subunit p22phox in heart, lung and kidney. The tissue antioxidant activity in lung, liver and kidney was decreased in diabetic rats, while it was increased in heart tissue. NAC reduced the expression of p22phox and p67phox, suppressed p67phox membrane translocation, and reduced free 15-F2t-isoprostane levels in all tissues. NAC increased antioxidant activity in liver and lung, but did not significantly affect antioxidant activity in heart and kidney. Conclusion The current study shows that NAC inhibits NADPH oxidase activation in diabetes and attenuates tissue oxidative damage in all organs, even though its effects on antioxidant activity are tissue specific. PMID:22318816

Lei, Shaoqing; Liu, Yanan; Liu, Huimin; Yu, Hong; Wang, Hui

2012-01-01

308

XJP-1 protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by inhibiting NADPH oxidase subunit expression and modulating the PI3K/Akt/eNOS pathway.  

PubMed

Endothelial apoptosis triggered by oxidized low-density lipoprotein (ox-LDL) can accelerate the progression of endothelial dysfunction in atherosclerosis. (±)7,8-Dihydroxy-3-methyl-isochromanone-4 (XJP-1) is a natural phenolic compound derived from banana peel. In the present study, we investigated the anti-apoptotic effect of XJP-1 in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL and explored underlying mechanisms. Our results showed that in the presence of ox-LDL, XJP-1 significantly attenuated ox-LDL-mediated cytotoxicity, apoptosis, caspase-3 activation, reactive oxygen species (ROS) generation, and NADPH oxidase subunit (p22phox and p47phox) expression in HUVECs. In addition, the anticytotoxic and anti-apoptotic effect of XJP-1 was partially inhibited by a PI3K inhibitor (LY294002), an Akt inhibitor (SH-6), a specific eNOS inhibitor (l-NAME) and a NADPH oxidase inhibitor (DPI). In exploring the underlying mechanisms of XJP-1 action, we found that XJP-1 eliminated ox-LDL-induced dephosphorylation of Akt and eNOS in a dose-dependent manner. However, XJP-1 alone upregulation of Akt and eNOS phosphorylation were blocked by LY294002 and SH-6. Moreover, XJP-1 increased NO production, but this effect was abolished by LY294002, SH-6 and l-NAME. The inhibition of ox-LDL-induced endothelial dysfunction by XJP-1 is due at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway. PMID:22980246

Fu, Rong; Wang, Qiujuan; Guo, Qinglong; Xu, Jinyi; Wu, Xiaoming

2013-01-01

309

A case-control study of rheumatoid arthritis identifies an associated single nucleotide polymorphism in the NCF4 gene, supporting a role for the NADPH-oxidase complex in autoimmunity  

Microsoft Academic Search

Rheumatoid arthritis (RA) is a chronic inflammatory disease with a heritability of 60%. Genetic contributions to RA are made by multiple genes, but only a few gene associations have yet been confirmed. By studying animal models, reduced capacity of the NADPH-oxidase (NOX) complex, caused by a single nucleotide polymorphism (SNP) in one of its components (the NCF1 gene), has been

Lina M Olsson; Anna-Karin Lindqvist; Henrik Källberg; Leonid Padyukov; Harald Burkhardt; Lars Alfredsson; Lars Klareskog; Rikard Holmdahl

2007-01-01

310

Naloxone inhibits immune cell function by suppressing superoxide production through a direct interaction with gp91phox subunit of NADPH oxidase  

PubMed Central

Background Both (-) and (+)-naloxone attenuate inflammation-mediated neurodegeneration by inhibition of microglial activation through superoxide reduction in an opioid receptor-independent manner. Multiple lines of evidence have documented a pivotal role of overactivated NADPH oxidase (NOX2) in inflammation-mediated neurodegeneration. We hypothesized that NOX2 might be a novel action site of naloxone to mediate its anti-inflammatory actions. Methods Inhibition of NOX-2-derived superoxide by (-) and (+)-naloxone was measured in lipopolysaccharide (LPS)-treated midbrain neuron-glia cultures and phorbol myristate acetate (PMA)-stimulated neutrophil membranes by measuring the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt (WST-1) or ferricytochrome c. Further, various ligand (3H-naloxone) binding assays were performed in wild type and gp91phox-/- neutrophils and transfected COS-7 and HEK293 cells. The translocation of cytosolic subunit p47phox to plasma membrane was assessed by western blot. Results Both (-) and (+)-naloxone equally inhibited LPS- and PMA-induced superoxide production with an IC50 of 1.96 and 2.52 ?M, respectively. Competitive binding of 3H-naloxone with cold (-) and (+)-naloxone in microglia showed equal potency with an IC50 of 2.73 and 1.57 ?M, respectively. 3H-Naloxone binding was elevated in COS-7 and HEK293 cells transfected with gp91phox; in contrast, reduced 3H-naloxone binding was found in neutrophils deficient in gp91phox or in the presence of a NOX2 inhibitor. The specificity and an increase in binding capacity of 3H-naloxone were further demonstrated by 1) an immunoprecipitation study using gp91phox antibody, and 2) activation of NOX2 by PMA. Finally, western blot studies showed that naloxone suppressed translocation of the cytosolic subunit p47phox to the membrane, leading to NOX2 inactivation. Conclusions Strong evidence is provided indicating that NOX2 is a non-opioid novel binding site for naloxone, which is critical in mediating its inhibitory effect on microglia overactivation and superoxide production. PMID:22340895

2012-01-01

311

Studies of Mitochondrial and Nonmitochondrial Sources Implicate Nicotinamide Adenine Dinucleotide Phosphate Oxidase(s) in the Increased Skeletal Muscle Superoxide Generation That Occurs During Contractile Activity  

PubMed Central

Abstract Aims The sources of cytosolic superoxide in skeletal muscle have not been defined. This study examined the subcellular sites that contribute to cytosolic superoxide in mature single muscle fibers at rest and during contractile activity. Results: Isolated fibers from mouse flexor digitorum brevis loaded with superoxide and nitric-oxide-sensitive fluorescent probes, specific pathway inhibitors and immunolocalization techniques were used to identify subcellular sites contributing to cytosolic superoxide. Treatment with the electron transport chain complex III inhibitor, antimycin A, but not the complex I inhibitor, rotenone, caused increased cytosolic superoxide through release from the mitochondrial intermembrane space via voltage-dependent anion or Bax channels, but inhibition of these channels did not affect contraction-induced increases in cytosolic superoxide. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors decreased cytosolic superoxide at rest and following contractions. Protein and mRNA expression of NADPH oxidase subunits was demonstrated in single fibers. NOX2, NOX4, and p22phox subunits localized to the sarcolemma and transverse tubules; NOX4 was additionally expressed in mitochondria. Regulatory p40phox and p67phox proteins were found in the cytoplasm of resting fibers, but following contractions, p40phox appeared to translocate to the sarcolemma. Innovation: Superoxide and other reactive oxygen species generated by skeletal muscle are important regulators of muscle force production and adaptations to contractions. This study has defined the relative contribution of mitochondrial and cytosolic sources of superoxide within the cytosol of single muscle fibers at rest and during contractions. Conclusion: Muscle mitochondria do not modulate cytosolic superoxide in skeletal muscle but NADPH oxidase is a major contributor both at rest and during contractions. Antioxid. Redox Signal. 18, 603–621. PMID:23050834

Sakellariou, Giorgos Konstantinos; Vasilaki, Aphrodite; Palomero, Jesus; Kayani, Anna; Zibrik, Lea; McArdle, Anne

2013-01-01

312

Effect of contraceptive steroids on monoamine oxidase activity  

PubMed Central

Cyclical variations in monoamine oxidase activity during the human menstrual cycle, specific to the endometrium and modified in women undergoing contraceptive steroid treatment, may reflect changes in hormonal environment. Treatment of rats with individual constituents of the contraceptive pill causes analogous changes: oestrogens inhibit and progestogens potentiate uterine monoamine oxidase activity. ImagesFig. 2Fig. 3

Southgate, Jennifer; Collins, G. G. S.; Pryse-Davies, J.; Sandler, M.

1969-01-01

313

Brief Communications Brain Monoamine Oxidase A Activity Predicts Trait  

E-print Network

Brief Communications Brain Monoamine Oxidase A Activity Predicts Trait Aggression Nelly Alia of this behavior. A potential neu- rochemical target is monoamine oxidase A (MAO A), an enzyme involved in metabolism of monoamines in brain and other or- gans (Shih and Thompson, 1999). MAO A catalytic activity re

Goldstein, Rita

314

The role of respiratory burst oxidase homologues in elicitor-induced stomatal closure and hypersensitive response in Nicotiana benthamiana  

Microsoft Academic Search

Active oxygen species (AOS) are central components of the defence reactions of plants against pathogens. Plant respiratory burst oxidase homologues (RBOH) of gp91phox, a plasma membrane protein of the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, play a prominent role in AOS production. The role of two RBOH from Nicotiana benthamiana, NbrbohA and NbrbohB that encode plant NADPH oxidase in

Huajian Zhang; Qin Fang; Zhengguang Zhang; Yuanchao Wang; Xiaobo Zheng

2010-01-01

315

Gibberellin oxidase activities in Bradyrhizobium japonicum bacteroids.  

PubMed

Bradyrhizobium japonicum bacteroids isolated from root nodules of soybean (Glycine max.) plants converted the gibberellin (GA) precursor [(14)C1]GA12 into several products identified by combined gas chromatography-mass spectrometry as [(14)C1]GA24, [(14)C1]GA9, [(14)C1]GA15, GA9 17-nor-16-one and unidentified products. The oxidation of GA12, catalyzed by the GA 20-oxidase, was present in symbiotic bacteroids from plants around flowering, but not in bacteroids from plants at either an early vegetative stage or at late growth stages. Expression of cps and ks genes, involved in ent-kaurene biosynthesis, was also demonstrated in bacteroids from soybean plants around flowering. Earlier precursors of the GA pathway, ent-[(14)C1]kaurenoic acid or [(14)C4]GA12-aldehyde, were efficiently utilized by B. japonicum bacteroids to give labelled GA9 plus intermediates partially oxidized at C-20, as well as GA9 17-nor-16-one and an unidentified product. No 3? or 13-hydroxylated [(14)C]GAs were detected in any of the incubations. Moreover the C19-GAs [(14)C1]GA4 or [(14)C1]GA20 were recovered unconverted upon incubation with the bacteroids which supports the absence of GA 3?-hydroxylase activity in B. japonicum. The bacterial 20-oxidase utilized the 13-hydroxylated substrates [(14)C1]GA53, [(14)C1]GA44 or [(14)C1]GA19, although with less efficiency than [(14)C1]GA12 to give [(14)C1]GA20 as final product, while the 3?-hydroxylated substrate [(14)C1]GA14 was converted to [(14)C1]GA4 to a very small extent. Endogenous GA9 and GA24 were identified by GC-MS in methanolic nodule extracts. These results suggest that B. japonicum bacteroids would synthesize GA9 under the symbiotic conditions present in soybean root nodules. PMID:24378220

Méndez, Constanza; Baginsky, Cecilia; Hedden, Peter; Gong, Fan; Carú, Margarita; Rojas, María Cecilia

2014-02-01

316

Superoxide anion production by NADPH oxidase plays a major role in erectile dysfunction in middle-aged rats: prevention by antioxidant therapy.  

PubMed

INTRODUCTION.: Prevalence of erectile dysfunction (ED) increases progressively with aging, but the ED pathophysiology at its early stages is still poorly investigated. AIM.: This study aimed to evaluate the functional and molecular alterations of erectile function at middle age, focusing on the contribution of oxidative stress in erectile tissue for the ED. METHODS.: Young (3.5-month) and middle-aged (10-month) male Wistar rats were used. Rat corpus cavernosum (RCC) was dissected free and mounted in 10-mL organ baths containing Krebs solution. Intracavernosal pressure (ICP) in anesthetized rats was evaluated. MAIN OUTCOME MEASURES.: Concentration-response curves to endothelium-dependent and endothelium-independent agents, as well as to electrical field stimulation (EFS), were obtained in RCC strips. Measurement of cyclic guanosine monophosphate (cGMP) and expressions of neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS), gp91(phox) and superoxide dismutase-1 (SOD-1) expressions in RCC were evaluated. RESULTS.: ICP was significantly reduced in middle-aged compared with young rats. RCC relaxations to acetylcholine (10(-8) to 10(-2) ?M), sodium nitroprusside (10(-8) to 10(-2) ?M), sildenafil (10(-9) to 10(-5) ?M), BAY 41-2272 (10(-9) to 10(-5) ?M), and EFS (4-32?Hz) were decreased in middle-aged group, which were nearly normalized by apocynin (NADPH oxidase inhibitor; 10(-4) ?M) or SOD (75?U/mL). Prolonged treatment with apocynin (85?mg/rat/day, 4 weeks) also restored the impaired relaxations in middle-aged rats. Relaxations to 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (8-Br-cGMP; 10(-8) to 3?×?10(-4) ?M) remained unchanged between groups. Basal and stimulated cGMP production were lower in middle-aged group, an effect fully restored by apocynin and SOD. Protein expression of nNOS and phosphorylated eNOS (p-eNOS) (Ser-1177) reduced, whereas gp(91phox) mRNA expression increased in RCC from middle-aged rats. CONCLUSIONS.: ED in middle-aged rats is associated with decreased NO bioavailability in erectile tissue due to upregulation of NADPH oxidase subunit gp91(phox) and downregulation of nNOS/p-eNOS. Antioxidant therapies may be a good pharmacological approach to prevent ED at its early stages. PMID:23347406

Silva, Fábio H; Mónica, Fabíola Z; Báu, Fernando R; Brugnerotto, Ana F; Priviero, Fernanda B M; Toque, Haroldo A; Antunes, Edson

2013-04-01

317

Alzheimer disease ?-amyloid activity mimics cholesterol oxidase  

PubMed Central

The abnormal accumulation of amyloid ?-peptide (A?) in the form of senile (or amyloid) plaques is one of the main characteristics of Alzheimer disease (AD). Both cholesterol and Cu2+ have been implicated in AD pathogenesis and plaque formation. A? binds Cu2+ with very high affinity, forming a redox-active complex that catalyzes H2O2 production from O2 and cholesterol. Here we show that A?:Cu2+ complexes oxidize cholesterol selectively at the C-3 hydroxyl group, catalytically producing 4-cholesten-3-one and therefore mimicking the activity of cholesterol oxidase, which is implicated in cardiovascular disease. A? toxicity in neuronal cultures correlated with this activity, which was inhibited by Cu2+ chelators including clioquinol. Cell death induced by staurosporine or H2O2 did not elevate 4-cholesten-3-one levels. Brain tissue from AD subjects had 98% more 4-cholesten-3-one than tissue from age-matched control subjects. We observed a similar increase in the brains of Tg2576 transgenic mice compared with nontransgenic littermates; the increase was inhibited by in vivo treatment with clioquinol, which suggests that brain A? accumulation elevates 4-cholesten-3-one levels in AD. Cu2+-mediated oxidation of cholesterol may be a pathogenic mechanism common to atherosclerosis and AD. PMID:16127459

Puglielli, Luigi; Friedlich, Avi L.; Setchell, Kenneth D.R.; Nagano, Seiichi; Opazo, Carlos; Cherny, Robert A.; Barnham, Kevin J.; Wade, John D.; Melov, Simon; Kovacs, Dora M.; Bush, Ashley I.

2005-01-01

318

NADPH-diaphorase activity in the vagal afferent pathway of the dogfish, Triakis scyllia  

Microsoft Academic Search

Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was examined in the cranial sensory ganglia and brainstem of the banded dogfish, Triakis scyllia. Positive neurons were found in the vagal sensory ganglion projecting to the coelomic organs, but not in those projecting to the gills or the lateral line organs. Nerve terminals in the vagal lobe were also positive. No positive neurons

Kengo Funakoshi; Tetsuo Kadota; Yoshitoshi Atobe; Richard C Goris; Reiji Kishida

1997-01-01

319

Aldehyde oxidase activity in fresh human skin.  

PubMed

Human aldehyde oxidase (AO) is a molybdoflavoenzyme that commonly oxidizes azaheterocycles in therapeutic drugs. Although high metabolic clearance by AO resulted in several drug failures, existing in vitro-in vivo correlations are often poor and the extrahepatic role of AO practically unknown. This study investigated enzymatic activity of AO in fresh human skin, the largest organ of the body, frequently exposed to therapeutic drugs and xenobiotics. Fresh, full-thickness human skin was obtained from 13 individual donors and assayed with two specific AO substrates: carbazeran and zoniporide. Human skin explants from all donors metabolized carbazeran to 4-hydroxycarbazeran and zoniporide to 2-oxo-zoniporide. Average rates of carbazeran and zoniporide hydroxylations were 1.301 and 0.164 pmol?mg skin(-1)?h(-1), resulting in 13 and 2% substrate turnover, respectively, after 24 hours of incubation with 10 ?M substrate. Hydroxylation activities for the two substrates were significantly correlated (r(2) = 0.769), with interindividual variability ranging from 3-fold (zoniporide) to 6-fold (carbazeran). Inclusion of hydralazine, an irreversible inhibitor of AO, resulted in concentration-dependent decrease of hydroxylation activities, exceeding 90% inhibition of carbazeran 4-hydroxylation at 100 ?M inhibitor. Reaction rates were linear up to 4 hours and well described by Michaelis-Menten enzyme kinetics. Comparison of carbazeran and zoniporide hydroxylation with rates of triclosan glucuronidation and sulfation and p-toluidine N-acetylation showed that cutaneous AO activity is comparable to tested phase II metabolic reactions, indicating a significant role of AO in cutaneous drug metabolism. To our best knowledge, this is the first report of AO enzymatic activity in human skin. PMID:25249692

Manevski, Nenad; Balavenkatraman, Kamal Kumar; Bertschi, Barbara; Swart, Piet; Walles, Markus; Camenisch, Gian; Schiller, Hilmar; Kretz, Olivier; Ling, Barbara; Wettstein, Reto; Schaefer, Dirk J; Pognan, Francois; Wolf, Armin; Litherland, Karine

2014-12-01

320

A COUPLED MICROSOMAL-ACTIVATING/EMBRYO CULTURE SYSTEM: TOXICITY OF REDUCED BETA-NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADPH)  

EPA Science Inventory

An NADPH-dependent microsomal-activating system has been coupled to a rat embryo culture in vitro. No embryonic morphological abnormalities or decrease in final yolk sac or embryo DNA and protein contents occurred when 0.2 mM NADPH was used in this coupled system. In contrast, 1....

321

The NADPH oxidase Nox2 regulates VEGFR1/CSF-1R-mediated microglial chemotaxis and promotes early postnatal infiltration of phagocytes in the subventricular zone of the mouse cerebral cortex.  

PubMed

The phagocyte NADPH oxidase Nox2 generates superoxide ions implicated in the elimination of microorganisms and the redox control of inflammatory signaling. However, the role of Nox2 in phagocyte functions unrelated to immunity or pathologies is unknown. During development, oriented cell migrations insure the timely recruitment and function of phagocytes in developing tissues. Here, we have addressed the role of Nox2 in the directional migration of microglial cells during development. We show that microglial Nox2 regulates the chemotaxis of purified microglia mediated by the colony stimulating factor-1 receptor (CSF-1R) and the vascular endothelial growth factor receptor-1 (VEGFR1). Stimulation of these receptors triggers activation of Nox2 at the leading edge of polarized cells. In the early postnatal stages of mouse brain development, Nox2 is activated in macrophages / microglial cells in the lateral ventricle or the adjacent subventricular zone (SVZ). Fluorescent microglia injected into the lateral ventricle infiltrate the dorso-caudal SVZ through a mechanism that is blocked by pretreatment of the injected cells with an irreversible Nox inhibitor. Infiltration of endogenous microglia into the caudal SVZ of the cerebral cortex is prevented by (1) Nox2 gene deficiency, (2) treatment with a Nox2 inhibitor (apocynin), and (3) invalidation of the VEGFR1 kinase. We conclude that phagocytes move out of the lateral ventricle soon after birth and infiltrate the cortical SVZ through a mechanism requiring microglial Nox2 and VEGFR1 activation. Nox2 therefore modulates the migration of microglia and their development. PMID:23836548

Lelli, Aurélia; Gervais, Annie; Colin, Catherine; Chéret, Cyril; Ruiz de Almodovar, Carmen; Carmeliet, Peter; Krause, Karl-Heinz; Boillée, Séverine; Mallat, Michel

2013-09-01

322

Expanded ataxin-7 cause toxicity by inducing ROS production from NADPH oxidase complexes in a stable inducible Spinocerebellar ataxia type 7 (SCA7) model  

PubMed Central

Background Spinocerebellar ataxia type 7 (SCA7) is one of nine inherited neurodegenerative disorders caused by polyglutamine (polyQ) expansions. Common mechanisms of disease pathogenesis suggested for polyQ disorders include aggregation of the polyQ protein and induction of oxidative stress. However, the exact mechanism(s) of toxicity is still unclear. Results In this study we show that expression of polyQ expanded ATXN7 in a novel stable inducible cell model first results in a concomitant increase in ROS levels and aggregation of the disease protein and later cellular toxicity. The increase in ROS could be completely prevented by inhibition of NADPH oxidase (NOX) complexes suggesting that ATXN7 directly or indirectly causes oxidative stress by increasing superoxide anion production from these complexes. Moreover, we could observe that induction of mutant ATXN7 leads to a decrease in the levels of catalase, a key enzyme in detoxifying hydrogen peroxide produced from dismutation of superoxide anions. This could also contribute to the generation of oxidative stress. Most importantly, we found that treatment with a general anti-oxidant or inhibitors of NOX complexes reduced both the aggregation and toxicity of mutant ATXN7. In contrast, ATXN7 aggregation was aggravated by treatments promoting oxidative stress. Conclusion Our results demonstrates that oxidative stress contributes to ATXN7 aggregation as well as toxicity and show that anti-oxidants or NOX inhibition can ameliorate mutant ATXN7 toxicity. PMID:22827889

2012-01-01

323

TNF-{alpha} upregulates the A{sub 2B} adenosine receptor gene: The role of NAD(P)H oxidase 4  

SciTech Connect

Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-{alpha}) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A{sub 2B} adenosine receptor (A{sub 2B}AR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-{alpha}. Here, we show a regulatory loop by which TNF-{alpha} upregulates the A{sub 2B}AR in VSMC in vitro and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A{sub 2B}AR, and Nox inhibitors dampen the effect of TNF-{alpha}. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A{sub 2B}AR.

St Hilaire, Cynthia; Koupenova, Milka; Carroll, Shannon H.; Smith, Barbara D. [Department of Biochemistry, Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118 (United States); Ravid, Katya [Department of Biochemistry, Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118 (United States)], E-mail: ravid@biochem.bumc.bu.edu

2008-10-24

324

NADPH Oxidase NOX2 Defines a New Antagonistic Role for Reactive Oxygen Species and cAMP/PKA in the Regulation of Insulin Secretion  

PubMed Central

In insulin-secreting cells, expression of NADPH oxidase (NOX), a potent source of ROS, has been reported, along with controversial findings regarding its function. Here, the role of NOXs was investigated: first by expression and cellular localization in mouse and human pancreatic islets, and then by functional studies in islets isolated from Nox isoform–specific knockout mice. Both human and mouse ?-cells express NOX, in particular NOX2. With use of Nox isoform–specific knockout mice, functional analysis revealed Nox2 as the predominant isoform. In human islets, NOX2 colocalized with both insulin granules and endosome/lysosome membranes. Nox2-deficient islets stimulated with 22.8 mmol/L glucose exhibited potentiation of insulin release compared with controls, an effect confirmed with in vitro knockdown of Nox2. The enhanced secretory function in Nox2-deficient islets was associated with both lower superoxide levels and elevated cAMP concentrations. In control islets, GLP-1 and other cAMP inducers suppressed glucose-induced ROS production similarly to Nox2 deficiency. Inhibiting cAMP-dependent protein kinase reduced the secretory response in Nox2-null islets, although not in control islets. This study ascribes a new role for NOX2 in pancreatic ?-cells as negative modulator of the secretory response, reducing cAMP/PKA signaling secondary to ROS generation. Results also show reciprocal inhibition between the cAMP/PKA pathway and ROS. PMID:22933115

Li, Ning; Li, Bin; Brun, Thierry; Deffert-Delbouille, Christine; Mahiout, Zahia; Daali, Youssef; Ma, Xiao-Juan; Krause, Karl-Heinz; Maechler, Pierre

2012-01-01

325

Platelet phenolsulphotransferase activity, monoamine oxidase activity and peripheral-type benzodiazepine binding in demented patients  

Microsoft Academic Search

Summary Blood platelet phenolsulphotransferase and monoamine oxidase activities, as well as platelet peripheral-type benzodiazepine binding have been studied in several neuropsychiatric disorders, in order to identify biochemical markers for altered brain functioning. In the present work, we determined platelet phenolsulphotransferase and monoamine oxidase activities in demented patients: they showed significantly higher phenolsulphotransferase and monoamine oxidase activities than controls. A significant

P. Bongioanni; M. Donato; M. Castagna; F. Gemignani

1996-01-01

326

Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis  

PubMed Central

Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability. PMID:24113189

Wiel, C; Augert, A; Vincent, D F; Gitenay, D; Vindrieux, D; Le Calve, B; Arfi, V; Lallet-Daher, H; Reynaud, C; Treilleux, I; Bartholin, L; Lelievre, E; Bernard, D

2013-01-01

327

Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis.  

PubMed

Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability. PMID:24113189

Wiel, C; Augert, A; Vincent, D F; Gitenay, D; Vindrieux, D; Le Calvé, B; Arfi, V; Lallet-Daher, H; Reynaud, C; Treilleux, I; Bartholin, L; Lelievre, E; Bernard, D

2013-01-01

328

NADPH oxidase subunit p22(phox)-mediated reactive oxygen species contribute to angiogenesis and tumor growth through AKT and ERK1/2 signaling pathways in prostate cancer.  

PubMed

Excessive generation of reactive oxygen species (ROS) in cancer cells is associated with cancer development, but the underlying mechanisms and therapeutic significance remain elusive. In this study, we reported that levels of ROS and p22(phox) expression are greatly increased in human prostate cancer tissues, and knockdown of p22(phox) by specific small interfering RNA (siRNA) decreased ROS levels in prostate cancer cells. We also showed that stable downregulation of p22(phox) in prostate cancer cells inhibited cell proliferation and colony formation, which was mediated by AKT and extracellular signal-regulated kinase (ERK)1/2 signaling pathways and their downstream molecules hypoxia-inducible factor 1? (HIF-1?) and vascular endothelial growth factor (VEGF). The NADPH oxidase subunit NOX1 was also elevated in prostate cancer cells, and was involved in activation of AKT/ERK/HIF-1/VEGF pathway and regulation of cell proliferation. Knockdown of p22(phox) resulted in inhibition of tumor angiogenesis and tumor growth in nude mice. These findings reveal a new function of p22(phox) in tumor angiogenesis and tumor growth, and suggest that p22(phox) is a potential novel target for prostate cancer treatment. PMID:24113386

Li, Qi; Fu, Guang-Bo; Zheng, Ji-Tai; He, Jun; Niu, Xiao-Bing; Chen, Qiu-Dan; Yin, Yu; Qian, Xu; Xu, Qing; Wang, Min; Sun, An-Fang; Shu, Yongqian; Rui, Hallgeir; Liu, Ling-Zhi; Jiang, Bing-Hua

2013-12-01

329

Caffeine metabolism in cystic fibrosis: Enhanced xanthine oxidase activity  

Microsoft Academic Search

Study objective: To characterize the activities of the P450 mixed-function oxidase CYP1A2 as well as the cytosolic enzymes N-acetyltransferase and xanthine oxidase using caffeine as a probe in children with cystic fibrosis compared to age-matched healthy control subjects.Methods: After administration of caffeine (cola beverage) to 12 children with cystic fibrosis (age range, 5 to 11 years) and 12 healthy control

Bettina A Hamelin; Keyi Xu; François Vallé; Luc Manseau; Monique Richer; Marc LeBel; Marc LeBel PharmD

1994-01-01

330

Alteration of inner-membrane components and damage to electron-transfer activities of bovine heart submitochondrial particles induced by NADPH-dependent lipid peroxidation.  

PubMed Central

We investigated the changes of the inner-membrane components and the electron-transfer activities of bovine heart submitochondrial particles induced by the lipid peroxidation supported by NADPH in the presence of ADP-Fe3+. Most of the polyunsaturated fatty acids were lost as a result of the peroxidation, and phospholipids were changed to polar species. Ubiquinone was also modified to polar substances as the peroxidation proceeded. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed the disappearance of 27000-Mr and 30000-Mr proteins and the appearance of highly polymerized substances. Flavins and cytochromes were not diminished, but the respiratory activity was lost. The reactions of NADH oxidase and NADH-cytochrome c reductase were most sensitive to the peroxidation, followed by those of succinate oxidase and succinate-cytochrome c reductase. Succinate dehydrogenase and duroquinol-cytochrome c reductase were inactivated by more extensive peroxidation, but cytochrome c oxidase was only partially inactivated. NADH-ferricyanide reductase was not inactivated. The pattern of the inactivation indicated that the lipid peroxidation affected the electron transport intensively between NADH dehydrogenase and ubiquinone, and moderately at the succinate dehydrogenase step and between ubiquinone and cytochrome c. Images Fig. 2. PMID:7082319

Narabayashi, H; Takeshige, K; Minakami, S

1982-01-01

331

NADPH-diaphorase activity in the digestive system of gastropod molluscs Achatina fulica and Littorina littorea  

Microsoft Academic Search

Distribution and morphological peculiarities of nitroxidergic elements throughout the entire length of digestive tract was\\u000a studied for the first time in gastropod molluscs Littorina littorea (Prosobranchia) and Achatina fulica (Pulmonata) using histochemical detection of NADPH-diaphorase (NADPHd). NO-ergic cells and fibers were revealed in all parts\\u000a of the mollusc digestive system beginning from esophagus. Intensive NADPHd activity is found in a

O. V. Zaitseva; T. V. Kuznetsova; T. G. Markosova

2009-01-01

332

Evidence for the involvement of NADPH oxidase in adenosine receptor-mediated control of coronary flow using A1 and A3 knockout mice  

PubMed Central

The NADPH oxidase (Nox) subunits 1, 2 (gp91 phox), and 4 are the major sources for reactive oxygen species (ROS) in cardiovascular system. In conditions such as ischemia–reperfusion injury, and hypoxia, both ROS and adenosine are released suggesting a possible interaction. We hypothesized that ROS generated through Nox is involved in adenosine-induced coronary flow (CF) responses. Adenosine (10?8–10?5.5 mol/L) increased CF in isolated hearts from wild-type (WT; C57BL/6), A1 adenosine receptor (AR) knockout (A1KO), A3AR KO (A3KO) and A1 and A3AR double KO (A1/A3DKO) mice. The Nox inhibitors apocynin (10?5 mol/L) and gp91 ds-tat (10?6 mol/L) or the superoxide dismutase (SOD) and catalase-mimicking agent EUK134 (50 ?mol/L) decreased the adenosine-enhanced CF in the WT and all the KOs. Additionally, adenosine increased phosphorylation of p47-phox subunit and extracellular signal-regulated kinase (ERK) 1/2 without changing protein expression of Nox isoforms in WT. Moreover, intracellular superoxide production was increased by adenosine and CGS-21680 (a selective A2A agonist), but not BAY 60-6583 (a selective A2B agonist), in mouse coronary artery smooth muscle cells (CASMCs) and endothelial cells (CAECs). This superoxide increase was inhibited by the gp91 ds-tat and ERK 1/2 inhibitor (PD98059). In conclusion, adenosine-induced increase in CF in isolated heart involves Nox2-generated superoxide, possibly through ERK 1/2 phosphorylation with subsequent p47-phox subunit phosphorylation. This adenosine/Nox/ROS interaction occurs in both CASMCs and CAECs, and involves neither A1 nor A3 ARs, but possibly A2A ARs in mouse. PMID:24159377

El-Awady, Mohammed S; Rajamani, Uthra; Teng, Bunyen; Tilley, Stephen L; Mustafa, S Jamal

2013-01-01

333

Combined NADPH Oxidase 1 and Interleukin 10 Deficiency Induces Chronic Endoplasmic Reticulum Stress and Causes Ulcerative Colitis-Like Disease in Mice  

PubMed Central

Ulcerative colitis (UC) is a chronic inflammatory bowel disease affecting the rectum which progressively extents. Its etiology remains unknown and the number of treatments available is limited. Studies of UC patients have identified an unbalanced endoplasmic reticulum (ER) stress in the non-inflamed colonic mucosa. Animal models with impaired ER stress are sensitive to intestinal inflammation, suggesting that an unbalanced ER stress could cause inflammation. However, there are no ER stress-regulating strategies proposed in the management of UC partly because of the lack of relevant preclinical model mimicking the disease. Here we generated the IL10/Nox1dKO mouse model which combines immune dysfunction (IL-10 deficiency) and abnormal epithelium (NADPH oxidase 1 (Nox1) deficiency) and spontaneously develops a UC-like phenotype with similar complications (colorectal cancer) than UC. Our data identified an unanticipated combined role of IL10 and Nox1 in the fine-tuning of ER stress responses in goblet cells. As in humans, the ER stress was unbalanced in mice with decreased eIF2? phosphorylation preceding inflammation. In IL10/Nox1dKO mice, salubrinal preserved eIF2? phosphorylation through inhibition of the regulatory subunit of the protein phosphatase 1 PP1R15A/GADD34 and prevented colitis. Thus, this new experimental model highlighted the central role of epithelial ER stress abnormalities in the development of colitis and defined the defective eIF2? pathway as a key pathophysiological target for UC. Therefore, specific regulators able to restore the defective eIF2? pathway could lead to the molecular remission needed to treat UC. PMID:25014110

Tréton, Xavier; Pedruzzi, Eric; Guichard, Cécile; Ladeiro, Yannick; Sedghi, Shirin; Vallée, Mélissa; Fernandez, Neike; Bruyčre, Emilie; Woerther, Paul-Louis; Ducroc, Robert; Montcuquet, Nicolas; Freund, Jean-Noel; Van Seuningen, Isabelle; Barreau, Frédérick; Marah, Assiya; Hugot, Jean-Pierre; Cazals-Hatem, Dominique; Bouhnik, Yoram; Daniel, Fanny; Ogier-Denis, Eric

2014-01-01

334

Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism  

PubMed Central

Background Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1st and 2nd EF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants. Results The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca2+ binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 105 and 5.0 × 102 M-1 with ?Hs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1st non-canonical EF-hand is largely enhanced by the binding of Ca2+ to the 2nd canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca2+ binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its ?-helix secondary structure content. All measurements exclude Mg2+-binding in NCaBD. Conclusions We demonstrated that the 1st non-canonical EF-hand of NOX5 has very weak Ca2+ binding affinity compared with the 2nd canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca2+ binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca2+ specific. Graphical abstract PMID:22490336

2012-01-01

335

A novel biofuel cell harvesting energy from activated human macrophages  

Microsoft Academic Search

Macrophage phagocytosis activates NADPH oxidase, an electron transport system in the plasma membrane. This study examined the feasibility of utilizing electrons transferred through the plasma membrane via NADPH oxidase to run a biofuel cell. THP-1 human monocytic cells were chemically stimulated to differentiate into macrophages. Further they were activated to induce a phagocytic response. During differentiation, cells became adherent to

Miho Sakai; Andreas Vonderheit; Xun Wei; Claudia Küttel; Andreas Stemmer

2009-01-01

336

Synergistic effect of NADH on NADPH-dependent acetaminophen activation in liver microsomes and its inhibition by cyanide  

Microsoft Academic Search

The effects of NADH and cyanide on NADPH-dependent acetaminophen activation in rat and mouse liver microsomes were studied. In both rat and mouse microsomes, NADPH-dependent acetaminophen-glutathione conjugate production was synergistically enhanced by the addition of NADH, whereas NADH alone did not initiate this reaction. The data suggest that the second electron in this reaction may be transferred from NADH. The

Chifumi Sato; Fumiaki Marumo

1991-01-01

337

CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity  

PubMed Central

Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10?6±0.21 M·min?1 and 0.32±0.02 s?1, respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal. PMID:23577125

Su, Jianmei; Bao, Peng; Bai, Tenglong; Deng, Lin; Wu, Hui; Liu, Fan; He, Jin

2013-01-01

338

CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity.  

PubMed

Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10(-6)±0.21 M·min(-1) and 0.32±0.02 s(-1), respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal. PMID:23577125

Su, Jianmei; Bao, Peng; Bai, Tenglong; Deng, Lin; Wu, Hui; Liu, Fan; He, Jin

2013-01-01

339

Angiotensin II (AT1) Receptors and NADPH Oxidase Regulate Cl Current Elicited by  1 Integrin Stretch in Rabbit Ventricular Myocytes  

Microsoft Academic Search

Direct stretch of ? 1 integrin activates an outwardly rectifying, tamoxifen-sensitive Clcurrent (ClSAC) via focal adhesion kinase (FAK) and\\/or Src. The characteristics of ClSAC resemble those of the volume-sensitive Clcurrent, I Cl,swell . Because myocyte stretch releases angiotensin II (AngII), which binds AT1 receptors (AT1R) and stimulates FAK and Src in an autocrine-paracrine loop, we tested whether AT1R and their

David M. Browe; Clive M. Baumgarten

2004-01-01

340

Deletion of P399{sub E}401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency  

SciTech Connect

Highlights: {yields} Mutations in human POR cause congenital adrenal hyperplasia. {yields} We are reporting a novel 3 amino acid deletion mutation in POR P399{sub E}401del. {yields} POR mutation P399{sub E}401del decreased P450 activities by 60-85%. {yields} Impairment of steroid metabolism may be caused by multiple hits. {yields} Severity of aromatase inhibition is related to degree of in utero virilization. -- Abstract: P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399{sub E}401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399{sub E}401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17{alpha}-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399{sub E}401 revealed reduced stability and flexibility of the mutant. In conclusion, P399{sub E}401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399{sub E}401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

Flueck, Christa E., E-mail: christa.flueck@dkf.unibe.ch [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland); Mallet, Delphine [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France)] [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France); Hofer, Gaby [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland)] [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland); Samara-Boustani, Dinane [Hopital Necker-Enfants malades, Paris (France)] [Hopital Necker-Enfants malades, Paris (France); Leger, Juliane [Hopital Robert Debre, Paris (France)] [Hopital Robert Debre, Paris (France); Polak, Michel [Hopital Necker-Enfants malades, Paris (France)] [Hopital Necker-Enfants malades, Paris (France); Morel, Yves [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France)] [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France); Pandey, Amit V., E-mail: amit@pandeylab.org [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland)

2011-09-09

341

Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency.  

PubMed

P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399_E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399_E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17?-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399_E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399_E401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399_E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase. PMID:21843508

Flück, Christa E; Mallet, Delphine; Hofer, Gaby; Samara-Boustani, Dinane; Leger, Juliane; Polak, Michel; Morel, Yves; Pandey, Amit V

2011-09-01

342

Chronic monoamine oxidase-B inhibitor treatment blocks monoamine oxidase-A enzyme activity.  

PubMed

Patients with Parkinson's disease receive selective irreversible monoamine oxidase (MAO)-B inhibitors, but their effects on MAO-A activity are not known during long-term application. We determined MAO-A inhibition in plasma samples from patients with MAO-B inhibitor intake or without MAO-B inhibitor treatment and from healthy controls. We detected a 70 % reduction of MAO-A activity in patients with MAO-B inhibitor therapy in comparison to the other groups. Our results suggest that treatment with MAO-B inhibitor may also influence MAO-A activity in vivo, when administered daily. PMID:24272680

Bartl, Jasmin; Müller, Thomas; Grünblatt, Edna; Gerlach, Manfred; Riederer, Peter

2014-04-01