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1

Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components.  

PubMed

Activation of the superoxide-generating NADPH oxidase system of human neutrophils involves the assembly of several neutrophil components, some located on the plasma membrane and others in the cytosol. It has recently been established that one of the required components for NADPH oxidase activity is the GTP-binding protein Rac. To further investigate the role of Rac in the NADPH oxidase system, studies were carried out to determine its subcellular distribution in resting and activated human neutrophils. In resting cells, Rac and an associated guanine nucleotide regulatory factor, GDP dissociation inhibitor (GDI), were located only in the cytosol, along with other known oxidase factors, p47-phox and p67-phox. After activation of neutrophils with phorbol 12-myristate 13-acetate or formyl-methionyl-leucyl-phenylalanine, Rac was translocated from the cytosol to the plasma membrane, and this translocation corresponded temporally with the translocation of p47-phox and p67-phox and with the generation of superoxide. GDI remained localized to the cytosol, suggesting activation of the oxidase involved dissociation of the Rac-GDI complex prior to Rac translocation. Determination of the quantities of cytosolic factors associated with the plasma membrane indicated that Rac, p47-phox, and p67-phox are translocated to the plasma membrane simultaneously in equimolar amounts, but that the membrane-associated cytochrome b was present at 3-4-fold molar excess. These findings suggest that Rac may play a role in assembly of the active NADPH oxidase complex. PMID:8407934

Quinn, M T; Evans, T; Loetterle, L R; Jesaitis, A J; Bokoch, G M

1993-10-01

2

Rosuvastatin reduces platelet recruitment by inhibiting NADPH oxidase activation.  

PubMed

Rosuvastatin increased vascular endothelial NO and attenuated platelet activation after ischemia-reperfusion in mice; nevertheless, the influence of rosuvastatin on the activation of human platelets and the underlying mechanism has never been investigated. In an in vitro study platelets from 8 healthy donors were incubated with scalar concentrations of rosuvastatin (1-10 ?M) before activation. Platelet recruitment (PR), that mimics the propagation of platelet aggregation and is dependent upon isoprostane formation, was investigated. PR was inhibited by rosuvastatin in concentration-dependent manner concomitantly with down-regulation of platelet release of the pro-thrombotic molecule CD40L. This effect was associated with lower production of platelet reactive oxygen species (ROS), isoprostane and activation of the glycoprotein IIb/IIIa and was counteracted by exogenous addition of isoprostanes. Conversely, rosuvastatin concentration-dependently increased platelet NO. Platelet isoprostane formation mainly depends from NADPH oxidase. Rosuvastatin concentration-dependently inhibited platelet sNOX2-dp release, a specific marker of NADPH oxidase activation, PKC phosphorylation and p47(phox) translocation from cytosol to membranes. In an ex vivo study 10 hypercolesterolemic patients were randomly allocated to diet or rosuvastatin (20 mg). We observed that as early as 2h after rosuvastatin PR, platelet isoprostanes formation, platelet CD40L and sNOX2-dp decreased while platelet NO increased; no changes were detected in diet-assigned patients. This study shows that in vitro rosuvastatin impairs platelet activation via inhibition of NOX2-derived oxidative stress. This effect, which is associated ex vivo with acute inhibition of platelet activation, suggests that rosuvastatin behaves as an antiplatelet drug. PMID:23022230

Pignatelli, Pasquale; Carnevale, Roberto; Di Santo, Serena; Bartimoccia, Simona; Nocella, Cristina; Vicario, Tommasa; Loffredo, Lorenzo; Angelico, Francesco; Violi, Francesco

2012-09-26

3

Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils  

Microsoft Academic Search

The gp91phox subunit of flavocytochrome b558 is the catalytic core of the phagocyte plasma membrane NADPH oxidase. Its activation occurs within lipid rafts and requires translocation of four subunits to flavocytochrome b558. gp91phox is the only glycosylated subunit of NADPH oxidase and no data exist about the structure or function of its glycans. Glycans, however, bind to lectins and this

Irina V. Gorudko; Ann V. Mukhortava; Brendan Caraher; Melody Ren; Sergey N. Cherenkevich; Gregory M. Kelly; Alexander V. Timoshenko

4

Molecular Basis of Phosphorylation-Induced Activation of the NADPH Oxidase  

Microsoft Academic Search

The multi-subunit NADPH oxidase complex plays a crucial role in host defense against microbial infection through the production of reactive oxygen species. Activation of the NADPH oxidase requires the targeting of a cytoplasmic p40-p47-p67phox complex to the membrane bound heterodimeric p22-gp91phox flavocytochrome. This interaction is prevented in the resting state due to an auto-inhibited conformation of p47phox. The X-ray structure

Yvonne Groemping; Karine Lapouge; Stephen J. Smerdon; Katrin Rittinger

2003-01-01

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

NADPH Oxidase and Neurodegeneration  

PubMed Central

NADPH oxidase (Nox) is a unique, multi-protein, electron transport system that produces large amounts of superoxide via the reduction of molecular oxygen. Nox-derived reactive oxygen species (ROS) are known to be involved in a variety of physiological processes, including host defense and signal transduction. However, over the past decade, the involvement of (Nox)-dependent oxidative stress in the pathophysiology of several neurodegenerative diseases has been increasingly recognized. ROS produced by Nox proteins contribute to neurodegenerative diseases through distinct mechanisms, such as oxidation of DNA, proteins, lipids, amino acids and metals, in addition to activation of redox-sensitive signaling pathways. In this review, we discuss the recent literature on Nox involvement in neurodegeneration, focusing on Parkinson and Alzheimer diseases.

Hernandes, Marina S; Britto, Luiz R G

2012-01-01

7

NADPH oxidase activity is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation  

PubMed Central

Phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is a form of spinal, serotonin-dependent synaptic plasticity that requires reactive oxygen species (ROS) formation. We tested the hypothesis that spinal NADPH oxidase activity is a necessary source of ROS for pLTF. Sixty minutes post-AIH (three 5-min episodes of 11% O2, 5 min intervals), integrated phrenic and hypoglossal (XII) nerve burst amplitudes were increased from baseline, indicative of phrenic and XII LTF. Intrathecal injections (?C4) of apocynin or diphenyleneiodonium chloride (DPI), two structurally and functionally distinct inhibitors of the NADPH oxidase complex, attenuated phrenic, but not XII, LTF. Immunoblots from soluble (cytosolic) and particulate (membrane) fractions of ventral C4 spinal segments revealed predominantly membrane localization of the NADPH oxidase catalytic subunit, gp91phox, whereas membrane and cytosolic expression were both observed for the regulatory subunits, p47phox and RAC1. Immunohistochemical analysis of fixed tissues revealed these same subunits in presumptive phrenic motoneurons of the C4 ventral horn, but not in neighbouring astrocytes or microglia. Collectively, these data demonstrate that NADPH oxidase subunits localized within presumptive phrenic motoneurons are a major source of ROS necessary for AIH-induced pLTF. Thus, NADPH oxidase activity is a key regulator of spinal synaptic plasticity, and may be a useful pharmaceutical target in developing therapeutic strategies for respiratory insufficiency in patients with, for example, cervical spinal injury.

MacFarlane, P M; Satriotomo, I; Windelborn, J A; Mitchell, G S

2009-01-01

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.

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

2012-01-01

9

Riboflavin kinase couples TNF receptor 1 to NADPH oxidase  

Microsoft Academic Search

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a

Benjamin Yazdanpanah; Katja Wiegmann; Vladimir Tchikov; Oleg Krut; Carola Pongratz; Michael Schramm; Andre Kleinridders; Thomas Wunderlich; Hamid Kashkar; Olaf Utermöhlen; Jens C. Brüning; Stefan Schütze; Martin Krönke

2009-01-01

10

NADPH-oxidase expression and in situ production of superoxide by osteoclasts actively resorbing bone  

Microsoft Academic Search

Recent reports have suggested that produc- tion of superoxide or other reactive oxygen species by activated osteoclasts may play a role in the complex process of bone resorption; however, the enzyme responsible for production of superoxide by osteoclasts has not been characterized. To determine if osteoclasts express NADPH-oxidase, a superoxide-generating en- zyme found in phagocytic leukocytes, immuno- histochemical studies were

Maria J. Steinbeck; William H. Appel; Arthur J. Verhoeven; Morris J. Karnovsky

1994-01-01

11

NADPH oxidase activation: a mechanism of erectile dysfunction in a rat model of sleep apnea.  

PubMed

Erectile dysfunction (ED) is a frequent occurrence in male patients with obstructive sleep apnea syndrome (OSAS). Long-term intermittent hypoxia (LTIH), one of the hallmarks of OSAS, could mediate ED. The objective of this study was to test the hypothesis that increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity contributes to ED in rat responses to LTIH. Healthy male Sprague-Dawley rats were randomly distributed into 4 groups: a LTIH group, an apocynin (a selective NADPH oxidase inhibitor)-treated LTIH group, a sham LTIH group, and an apocynin-treated sham group. Erectile function was examined by measuring the mean arterial blood pressure (MAP) and intracavernosal pressure (ICP) on electrical stimulation of the cavernous nerve. Real-time quantitative polymerase chain reaction and Western blot were used to examine mRNA and protein expression of NADPH oxidase subunit in corpus cavernosa (CC). The level of malondialdehyde and superoxide dismutase were detected by colorimetry. Nitric oxide synthase (NOS) isoforms in CC were also investigated. LTIH markedly attenuated the erectile responses (ICP/MAP), and these were partially prevented by apocynin treatment. Promoted oxidative stress-associated NADPH oxidase subunit activation was found in CC from LTIH rats. Decreased expression and activity of constitutive NOS (cNOS), including endothelial NOS and neuronal NOS, associated with enhanced inducible NOS (iNOS) expression and activity were observed in LTIH rats. Apocynin prevented the decrease in cNOS activity and inhibited iNOS expression and activity in LTIH rats. These results indicate that NADPH oxidase activation plays an important role in the pathogenesis of LTIH-mediated ED. PMID:22653964

Liu, Kui; Liu, Xian-Sheng; Xiao, Li; Shang, Jin; Li, Ming-Chao; Xu, Yong-Jian; Liu, Hui-Guo

2012-05-31

12

Attenuation of NADPH oxidase activation and glomerular filtration barrier remodeling with statin treatment.  

PubMed

Activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase by angiotensin II is integral to the formation of oxidative stress in the vasculature and the kidney. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition is associated with reductions of oxidative stress in the vasculature and kidney and associated decreases in albuminuria. Effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition on oxidative stress in the kidney and filtration barrier integrity are poorly understood. To investigate, we used transgenic TG(mRen2)27 (Ren2) rats, which harbor the mouse renin transgene and renin-angiotensin system activation, and an immortalized murine podocyte cell line. We treated young, male Ren2 and Sprague-Dawley rats with rosuvastatin (20 mg/kg IP) or placebo for 21 days. Compared with controls, we observed increases in systolic blood pressure, albuminuria, renal NADPH oxidase activity, and 3-nitrotryosine staining, with reductions in the rosuvastatin-treated Ren2. Structural changes on light and transmission electron microscopy, consistent with periarteriolar fibrosis and podocyte foot-process effacement, were attenuated with statin treatment. Nephrin expression was diminished in the Ren2 kidney and trended to normalize with statin treatment. Angiotensin II-dependent increases in podocyte NADPH oxidase activity and subunit expression (NOX2, NOX4, Rac, and p22(phox)) and reactive oxygen species generation were decreased after in vitro statin treatment. These data support a role for increased NADPH oxidase activity and subunit expression with resultant reactive oxygen species formation in the kidney and podocyte. Furthermore, statin attenuation of NADPH oxidase activation and reactive oxygen species formation in the kidney/podocyte seems to play roles in the abrogation of oxidative stress-induced filtration barrier injury and consequent albuminuria. PMID:18172055

Whaley-Connell, Adam; Habibi, Javad; Nistala, Ravi; Cooper, Shawna A; Karuparthi, Poorna R; Hayden, Melvin R; Rehmer, Nathan; DeMarco, Vincent G; Andresen, Bradley T; Wei, Yongzhong; Ferrario, Carlos; Sowers, James R

2008-01-02

13

Critical Role of NADPH Oxidase in Neuronal Oxidative Damage and Microglia Activation following Traumatic Brain Injury  

PubMed Central

Background Oxidative stress is known to play an important role in the pathology of traumatic brain injury. Mitochondria are thought to be the major source of the damaging reactive oxygen species (ROS) following TBI. However, recent work has revealed that the membrane, via the enzyme NADPH oxidase can also generate the superoxide radical (O2?), and thereby potentially contribute to the oxidative stress following TBI. The current study thus addressed the potential role of NADPH oxidase in TBI. Methodology/Principal Findings The results revealed that NADPH oxidase activity in the cerebral cortex and hippocampal CA1 region increases rapidly following controlled cortical impact in male mice, with an early peak at 1 h, followed by a secondary peak from 24–96 h after TBI. In situ localization using oxidized hydroethidine and the neuronal marker, NeuN, revealed that the O2? induction occurred in neurons at 1 h after TBI. Pre- or post-treatment with the NADPH oxidase inhibitor, apocynin markedly inhibited microglial activation and oxidative stress damage. Apocynin also attenuated TBI-induction of the Alzheimer's disease proteins ?-amyloid and amyloid precursor protein. Finally, both pre- and post-treatment of apocynin was also shown to induce significant neuroprotection against TBI. In addition, a NOX2-specific inhibitor, gp91ds-tat was also shown to exert neuroprotection against TBI. Conclusions/Significance As a whole, the study demonstrates that NADPH oxidase activity and superoxide production exhibit a biphasic elevation in the hippocampus and cortex following TBI, which contributes significantly to the pathology of TBI via mediation of oxidative stress damage, microglial activation, and AD protein induction in the brain following TBI.

Han, Dong; Nguyen, Khoi; Scott, Erin; Dong, Yan; Dhandapani, Krishnan M.; Brann, Darrell W.

2012-01-01

14

HSCARG Inhibits NADPH Oxidase Activity through Regulation of the Expression of p47phox  

PubMed Central

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase catalyzes the transfer of electrons from NADPH to O2, which is the main source of reactive oxygen species (ROS) in nonphagocytic cells. Excess ROS are toxic; therefore, keeping ROS in homeostasis in cells can protect cells from oxidative damage. It is meaningful to further understand the molecular mechanism by which ROS homeostasis is mediated. Human protein HSCARG is a newly identified oxidative sensor and a negative regulator of NF-?B. Here, we find that HSCARG represses the cellular ROS generation through inhibiting mRNA and protein expression of p47phox, a subunit of NADPH oxidase. In contrast, shRNA-mediated HSCARG knockdown increases endogenous p47phox expression level. And HSCARG has no obvious effect on ROS production in p47phox-depleted cells. Furthermore, HSCARG regulates p47phox through inhibition of NF-?B activity. Our findings identify HSCARG as a novel regulator in regulation of the activity of NADPH oxidase and ROS homeostasis.

Xiao, Weichun; Peng, Yanyan; Liu, Yong; Li, Zhi; Li, Senlin; Zheng, Xiaofeng

2013-01-01

15

Activation of the NADPH oxidase in a cell-free system from human neutrophils stimulated by phorbol myristate acetate  

SciTech Connect

Kinetics of activation of the NADPH oxidase in a fully soluble cell-free system from phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system in which Mg{sup 2+} and sodium dodecyl sulfate, an anionic detergent required for the activation of NADPH oxidase are contained, cytosol prepared from PMA-stimulated neutrophils failed to activate PMA-stimulated neutrophil oxidase. However, cytosol prepared from resting (control) neutrophils was capable of activating PMA-stimulated neutrophil oxidase in a cell-free system in which its Km for NADPH was almost similar to that of control neutrophil oxidase. Cytosol from PMA-stimulated neutrophils could not activate control neutrophil oxidase, although it did not contain any inhibitors of NADPH oxidase activation. These results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted, and that the affinity for NADPH of PMA-stimulated neutrophil oxidase may be the same as that of control neutrophil oxidase.

Umeki, Shigenobu (Kawasaki Medical School, Okayama (Japan))

1990-01-01

16

NADPH Oxidases and Angiotensin II Receptor Signaling  

PubMed Central

Over the last decade many studies have demonstrated the importance of reactive oxygen species (ROS) production by NADPH oxidases in angiotensin II (Ang II) signaling, as well as a role for ROS in the development of different diseases in which Ang II is a central component. In this review, we summarize the mechanism of activation of NADPH oxidases by Ang II and describe the molecular targets of ROS in Ang II signaling in the vasculature, kidney and brain. We also discuss the effects of genetic manipulation of NADPH oxidase function on the physiology and pathophysiology of the renin angiotensin system.

Garrido, Abel Martin; Griendling, Kathy K.

2010-01-01

17

Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation  

PubMed Central

S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b558; and (iii) to determine the S100A8 consensus site involved in cytochrome b558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase activation.

Berthier, Sylvie; Hograindleur, Marc-Andre; Paclet, Marie-Helene; Polack, Benoit; Morel, Francoise

2012-01-01

18

Superoxide anion is elevated in sympathetic neurons in DOCA-salt hypertension via activation of NADPH oxidase.  

PubMed

Superoxide anion (O2-*) production is elevated in sympathetic ganglion neurons and in the vasculature of hypertensive animals; however, it is not known what enzymatic pathway(s) are responsible for O2-* production. To determine the pathway(s) of O2-* production in sympathetic neurons, we examined the presence of mRNA of NADPH oxidase subunits in sympathetic ganglionic neurons and differentiated PC-12 cells. The mRNAs for NADPH oxidase subunits p47phox, p22phox, gp91phox, and NOX1 were present in sympathetic neurons and PC-12 cells, whereas the NOX4 homologue was present in sympathetic neurons but not PC-12 cells. Freshly dissociated celiac ganglion neurons from normal rats and PC-12 cells produced O2-* when treated with the PKC activator PMA; O2-* production increased by 317% and 254%, respectively. The PMA-evoked increases were reduced by pretreatment with the NADPH oxidase inhibitor apocynin. These findings indicate that NADPH oxidase is the primary source of O2-* in sympathetic ganglion neurons. When celiac ganglia from hypertensive rats were incubated with apocynin, O2-* levels were reduced to the same levels as normotensive animals, indicating that NADPH oxidase activity accounted for the elevated O2-* levels in hypertensive animals. To test this latter finding, we compared NADPH oxidase activity in extracts of prevertebral sympathetic ganglia of DOCA-salt hypertensive rats and sham-operated rats. NADPH oxidase activities were 49.9% and 78.6% higher in sympathetic ganglia of DOCA rats compared with normotensive controls when using beta-NADH and beta-NADPH as substrates, respectively. Thus elevated O2-* levels in hypertension may be a result of the increased activity of NADPH oxidase in postganglionic sympathetic neurons. PMID:16214837

Dai, Xiaoling; Cao, Xian; Kreulen, David L

2005-10-07

19

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

PubMed Central

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, we examine the effect of GT on the NADPH oxidase components in the cell-free activation assay. The O2?-generating ability of membranes obtained from GT-treated neutrophils is 40.0 and 30.6% lower, respectively, than the untreated counterparts when assayed with two distinct electron acceptors, suggesting that flavocytochrome b558 is affected in cells by GT. In contrast, the corresponding cytosol remains competent for activation. Next, GT addition in vitro to the assay consisting of flavocytochrome b558 and cytosolic components (native cytosol or recombinant p67phox, p47phox, and Rac2) causes a striking inhibition (50% inhibitory concentration = 3.3 ?M) when done prior to the stimulation with myristic acid. NADPH consumption is also prevented by GT, but the in vitro assembly of p67phox, p47phox, and Rac2 with flavocytochrome b558 is normal. Posterior addition of GT to the activated enzyme is ineffective. The separate treatment of membranes with GT also causes a marked loss of flavocytochrome b558's ability to reconstitute O2? generation, supporting the conclusion at the cellular level. The flavocytochrome b558 heme spectrum of the GT-treated membranes stays, however, unchanged, showing that hemes remain intact. These results suggest that GT directly harms site(s) crucial for electron transport in flavocytochrome b558, which is accessible only before oxidase activation.

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

2005-01-01

20

Epithelial-to-mesenchymal transition in podocytes mediated by activation of NADPH oxidase in hyperhomocysteinemia  

Microsoft Academic Search

The present study tested the hypothesis that hyperhomocysteinemia (hHcys) induces podocytes to undergo epithelial-to-mesenchymal\\u000a transition (EMT) through the activation of NADPH oxidase (Nox). It was found that increased homocysteine (Hcys) level suppressed\\u000a the expression of slit diaphragm-associated proteins, P-cadherin and zonula occludens-1 (ZO-1), in conditionally immortalized\\u000a mouse podocytes, indicating the loss of their epithelial features. Meanwhile, Hcys remarkably increased the

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

21

NADPH oxidase activity in preeclampsia with immortalized lymphoblasts used as models.  

PubMed

Upon activation, neutrophils release reactive oxygen species that are believed to contribute to the widespread manifestation of preeclampsia. Neutrophils have an NADPH oxidase enzyme that catalyzes the production of reactive oxygen species. Little is known about the manifestations of the activated response and the upstream signaling pathways that regulate this process in preeclampsia. It is hypothesized that genetic factors may contribute to the release of reactive oxygen species and consequently the pathophysiology of the disease. We used Epstein-Barr virus-immortalized lymphoblasts from third-trimester, preeclamptic, postpartum preeclamptic women and their respective control subjects to assess NADPH oxidase-mediated reactive oxygen species production by using luminol-derived chemiluminescence and dihydrorhodamine-123 fluorescence. There was no effect of pregnancy status on the lymphoblast phorbol ester-stimulated luminol chemiluminescence area under the curve. However, lymphoblasts from preeclamptic patients had significant elevation of the lymphoblast phorbol ester-stimulated luminol area under the curve (F statistic 10.922, P<0.002). Similar findings were evident with dihydrorhodamine-123. No differences were revealed between preeclamptic and control cells when measuring the abundance of the phox proteins using Western blotting. Studies with genistein and tyrphostin implicated tyrosine kinase-dependent mechanisms in the control of NADPH oxidase-associated increased reactive oxygen species production in preeclampsia. These data show that preeclampsia is associated with a predisposition to increased agonist-stimulated NADPH oxidase-mediated reactive oxygen species production. The enhancement of reactive oxygen species generation may be important in mediating the endothelial dysfunction seen in preeclampsia. PMID:12629036

Lee, Virginia M; Quinn, Paulene A; Jennings, Sonja C; Ng, Leong L

2003-03-10

22

Autophagy Protein Rubicon Mediates Phagocytic NADPH Oxidase Activation in Response to Microbial Infection or TLR Stimulation  

PubMed Central

Summary Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicon's actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection.

Yang, Chul-Su; Lee, Jong-Soo; Rodgers, Mary; Min, Chan-Ki; Lee, June-Yong; Kim, Hee Jin; Lee, Kwang-Hoon; Kim, Chul-Joong; Oh, Byungha; Zandi, Ebrahim; Yue, Zhenyu; Kramnik, Igor; Liang, Chengyu; Jung, Jae U.

2013-01-01

23

Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.  

PubMed

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22(phox), the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22(phox) is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase. PMID:19641494

Yazdanpanah, Benjamin; Wiegmann, Katja; Tchikov, Vladimir; Krut, Oleg; Pongratz, Carola; Schramm, Michael; Kleinridders, Andre; Wunderlich, Thomas; Kashkar, Hamid; Utermöhlen, Olaf; Brüning, Jens C; Schütze, Stefan; Krönke, Martin

2009-07-29

24

Mechanisms for the activation\\/electron transfer of neutrophil NADPH-oxidase complex and molecular pathology of chronic granulomatous disease  

Microsoft Academic Search

Summary Professional phagocytes, neutrophils, possess a unique membrane-associated NADPH-oxidase system, dormant in resting cells, which becomes activated upon exposure to the appropriate stimuli and catalyzes the one-electron reduction of molecular oxygen to superoxide, O2-. Oxidase activation involves the assembly, in the plasma membrane, of membrane-bound and cytosolic constituents of the oxidase system, which are disassembled in the resting state. The

S. Umeki

1994-01-01

25

Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase  

SciTech Connect

Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O{sub 2}{sup {center_dot}}{sup -} generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67{sup phox} siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91{sup phox} knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47{sup phox}, p67{sup phox} and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67{sup phox} siRNA. Exposure of MPMVEC obtained from gp91{sup phox} knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to activation of MAPKs through induced phosphorylation of p38 and ERK1/2 MAPKs that may further result in endothelial dysfunction through production of cytokines such as IL-6. Our results suggest that endothelial oxidative stress may be an important mechanism for PM-induced cardiovascular effects.

Mo Yiqun; Wan Rong [Department of Environmental Health and Occupational Health Sciences, School of Public Health and Information Sciences, University of Louisville, 485 E. Gray Street, Louisville, KY 40209 (United States); Chien Sufan [Department of Surgery, University of Louisville, Louisville, KY (United States); Tollerud, David J. [Department of Environmental Health and Occupational Health Sciences, School of Public Health and Information Sciences, University of Louisville, 485 E. Gray Street, Louisville, KY 40209 (United States); Zhang Qunwei [Department of Environmental Health and Occupational Health Sciences, School of Public Health and Information Sciences, University of Louisville, 485 E. Gray Street, Louisville, KY 40209 (United States)], E-mail: Qunwei.Zhang@louisville.edu

2009-04-15

26

Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase.  

PubMed Central

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins. Images

Sumimoto, H; Kage, Y; Nunoi, H; Sasaki, H; Nose, T; Fukumaki, Y; Ohno, M; Minakami, S; Takeshige, K

1994-01-01

27

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.

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

2012-01-01

28

SIRP? controls the activity of the phagocyte NADPH oxidase by restricting the expression of gp91(phox).  

PubMed

The phagocyte NADPH oxidase mediates oxidative microbial killing in granulocytes and macrophages. However, because the reactive oxygen species produced by the NADPH oxidase can also be toxic to the host, it is essential to control its activity. Little is known about the endogenous mechanism(s) that limits NADPH oxidase activity. Here, we demonstrate that the myeloid-inhibitory receptor SIRP? acts as a negative regulator of the phagocyte NADPH oxidase. Phagocytes isolated from SIRP? mutant mice were shown to have an enhanced respiratory burst. Furthermore, overexpression of SIRP? in human myeloid cells prevented respiratory burst activation. The inhibitory effect required interactions between SIRP? and its natural ligand, CD47, as well as signaling through the SIRP? cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Suppression of the respiratory burst by SIRP? was caused by a selective repression of gp91(phox) expression, the catalytic component of the phagocyte NADPH oxidase complex. Thus, SIRP? can limit gp91(phox) expression during myeloid development, thereby controlling the magnitude of the respiratory burst in phagocytes. PMID:23022485

van Beek, Ellen M; Zarate, Julian Alvarez; van Bruggen, Robin; Schornagel, Karin; Tool, Anton T J; Matozaki, Takashi; Kraal, Georg; Roos, Dirk; van den Berg, Timo K

2012-09-27

29

Differential activation of RAGE by HMGB1 modulates neutrophil-associated NADPH oxidase activity and bacterial killing  

PubMed Central

The receptor for advanced glycation end products (RAGE) plays an important role in host defense against bacterial infection. In the present experiments, we investigated the mechanisms by which RAGE contributes to the ability of neutrophils to eradicate bacteria. Wild-type (RAGE+/+) neutrophils demonstrated significantly greater ability to kill Eschericia coli compared with RAGE?/? neutrophils. After intraperitoneal injection of E. coli, increased numbers of bacteria were found in the peritoneal fluid from RAGE?/? as compared with RAGE+/+ mice. Exposure of neutrophils to the protypical RAGE ligand AGE resulted in activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and enhanced killing of E. coli, and intraperitoneal injection of AGE enhanced bacterial clearance during peritonitis. However, incubation of neutrophils with high mobility group box 1 protein (HMGB1), which also binds to RAGE, diminished E. coli-induced activation of NADPH oxidase in neutrophils and bacterial killing both in vitro and in vivo. Deletion of the COOH-terminal tail of HMGB1, a region necessary for binding to RAGE, abrogated the ability of HMGB1 to inhibit bacterial killing. Incubation of neutrophils with HMGB1 diminished bacterial or AGE-dependent activation of NADPH oxidase. The increase in phosphorylation of the p40phox subunit of NADPH oxidase that occurred after culture of neutrophils with E. coli was inhibited by exposure of the cells to HMGB1. These results showing that HMGB1, through RAGE-dependent mechanisms, diminishes bacterial killing by neutrophils as well as NADPH oxidase activation provide a novel mechanism by which HMGB1 can potentiate sepsis-associated organ dysfunction and mortality.

Tadie, Jean-Marc; Bae, Hong-Beom; Banerjee, Sami; Abraham, Edward

2012-01-01

30

NADPH oxidases in cardiovascular health and disease.  

PubMed

Increased oxidative stress plays an important role in the pathophysiology of cardiovascular diseases such as hypertension, atherosclerosis, diabetes, cardiac hypertrophy, heart failure, and ischemia-reperfusion. Although several sources of reactive oxygen species (ROS) may be involved, a family of NADPH oxidases appears to be especially important for redox signaling and may be amenable to specific therapeutic targeting. These include the prototypic Nox2 isoform-based NADPH oxidase, which was first characterized in neutrophils, as well as other NADPH oxidases such as Nox1 and Nox4. These Nox isoforms are expressed in a cell- and tissue-specific fashion, are subject to independent activation and regulation, and may subserve distinct functions. This article reviews the potential roles of NADPH oxidases in both cardiovascular physiological processes (such as the regulation of vascular tone and oxygen sensing) and pathophysiological processes such as endothelial dysfunction, inflammation, hypertrophy, apoptosis, migration, angiogenesis, and vascular and cardiac remodeling. The complexity of regulation of NADPH oxidases in these conditions may provide the possibility of targeted therapeutic manipulation in a cell-, tissue- and/or pathway-specific manner at appropriate points in the disease process. PMID:16771662

Cave, Alison C; Brewer, Alison C; Narayanapanicker, Anilkumar; Ray, Robin; Grieve, David J; Walker, Simon; Shah, Ajay M

31

Activation of mGluR5 and inhibition of NADPH oxidase improves functional recovery after traumatic brain injury.  

PubMed

Abstract Traumatic brain injury (TBI) induces microglial activation, which can contribute to secondary tissue loss. Activation of mGluR5 reduces microglial activation and inhibits microglial-mediated neurodegeneration in vitro, and is neuroprotective in experimental models of CNS injury. In vitro studies also suggest that the beneficial effects of mGluR5 activation involve nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibition in activated microglia. We hypothesized that activation of mGluR5 by the selective agonist CHPG after TBI in mice is neuroprotective and that its therapeutic actions are mediated by NADPH oxidase inhibition. Vehicle, CHPG, or CHPG plus the mGluR5 antagonist (MPEP), were administered centrally, 30 minutes post-TBI, and functional recovery and lesion volume was assessed. CHPG significantly attenuated post-traumatic sensorimotor and cognitive deficits, and reduced lesion volumes; these effects were blocked by MPEP, thereby indicating neuroprotection involved selective activation of mGluR5. CHPG treatment also reduced NF?B activity and nitrite production in lipopolysaccharide-stimulated microglia and the protective effects of CHPG treatment were abrogated in NADPH oxidase deficient microglial cultures (gp91(phox-/-)). To address whether the neuroprotective effects of CHPG are mediated via the inhibition of NADPH oxidase, we administered the NADPH oxidase inhibitor apocynin with or without CHPG treatment after TBI. Both apocynin or CHPG treatment alone improved sensorimotor deficits and reduced lesion volumes when compared with vehicle-treated mice; however, the combined CHPG + apocynin treatment was not superior to CHPG alone. These data suggest that the neuroprotective effects of activating mGluR5 receptors after TBI are mediated, in part, via the inhibition of NADPH oxidase. PMID:23199080

Loane, David J; Stoica, Bogdan A; Byrnes, Kimberly R; Jeong, William; Faden, Alan I

2013-01-30

32

Estrogen Attenuates Ischemic Oxidative Damage via an ER?-Mediated Inhibition of NADPH Oxidase Activation  

PubMed Central

The goal of this study was to elucidate the mechanisms of 17?-estradiol (E2) antioxidant and neuroprotective actions in stroke. The results reveal a novel extranuclear receptor-mediated antioxidant mechanism for E2 during stroke, as well as a hypersensitivity of the CA3/CA4 region to ischemic injury after prolonged hypoestrogenicity. E2 neuroprotection was shown to involve a profound attenuation of NADPH oxidase activation and superoxide production in hippocampal CA1 pyramidal neurons after stroke, an effect mediated by extranuclear ER?-mediated nongenomic signaling, involving Akt activation and subsequent phosphorylation/inactivation of Rac1, a factor critical for activation of NOX2 NADPH oxidase. Intriguingly, E2 nongenomic signaling, antioxidant action and neuroprotection in the CA1 region were lost after long-term E2 deprivation; and this loss was tissue-specific, as the uterus remained responsive to E2. Correspondingly, a remarkable loss of ER?, but not ER?, was observed in the CA1 following long-term E2 deprivation, with no change observed in the uterus. As a whole, the study reveals a novel, membrane-mediated antioxidant mechanism in neurons by E2, provides support and mechanistic insights for a “critical period” of E2 replacement in the hippocampus, and demonstrates a heretofore unknown hypersensitivity of the CA3/CA4 to ischemic injury after prolonged hypoestrogenicity.

Zhang, Quan-Guang; Raz, Limor; Wang, Ruimin; Han, Dong; De Sevilla, Liesl; Yang, Fang; Vadlamudi, Ratna K.; Brann, Darrell W.

2009-01-01

33

Chloride channels activated by swell can regulate the NADPH oxidase generated membrane depolarisation in activated human neutrophils  

SciTech Connect

Chloride channels activated by swell have important functions in many physiological processes. The phagocyte NADPH oxidase is essential for host defence and it generates superoxide by transferring electrons from the donor NADPH to the acceptor O{sub 2}. This electron current, induces a depolarisation of the plasma membrane. In this study, I report that chloride channels activated by swell can counteract the depolarisation induced by the NADPH oxidase. When a chloride conductance was activated by swelling, its inhibition by either 50 {mu}M NPPB or removing external chloride, depolarised the plasma membrane potential to +26 mV {+-} 3.1 (n = 4) and +40 {+-} 1 mV (n = 4), respectively. These channels were partially inhibited by the NADPH oxidase inhibitor AEBSF (1 mM) and potently inhibited by ZnCl{sub 2} (3 mM). These currents were not activated by a phosphorylation step and elevations in intracellular calcium did not appear to activate chloride currents similar to those activated by swell.

Ahluwalia, Jatinder [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom)], E-mail: j.ahluwalia@uel.ac.uk

2008-01-11

34

Leukotriene B(4) inhibits neutrophil apoptosis via NADPH oxidase activity: redox control of NF-?B pathway and mitochondrial stability.  

PubMed

Leukotriene B(4), an arachidonic acid-derived lipid mediator, is a known proinflammatory agent that has a direct effect upon neutrophil physiology, inducing reactive oxygen species generation by the NADPH oxidase complex and impairing neutrophil spontaneous apoptosis, which in turn may corroborate to the onset of chronic inflammation. Despite those facts, a direct link between inhibition of neutrophil spontaneous apoptosis and NADPH oxidase activation by leukotriene B(4) has not been addressed so far. In this study, we aim to elucidate the putative role of NADPH oxidase-derived reactive oxygen species in leukotriene B(4)-induced anti-apoptotic effect. Our results indicate that NADPH oxidase-derived reactive oxygen species are critical to leukotriene B(4) pro-survival effect on neutrophils. This effect also relies on redox modulation of nuclear factor kappaB signaling pathway. We have also observed that LTB(4)-induced Bad degradation and mitochondrial stability require NADPH oxidase activity. All together, our results strongly suggest that LTB(4)-induced anti-apoptotic effect in neutrophils occurs in a reactive oxygen species-dependent manner. We do believe that a better knowledge of the molecular mechanisms underlying neutrophil spontaneous apoptosis may contribute to the development of more successful strategies to control chronic inflammatory conditions such as rheumatoid arthritis. PMID:22884975

Barcellos-de-Souza, Pedro; Canetti, Cláudio; Barja-Fidalgo, Christina; Arruda, Maria Augusta

2012-08-04

35

Activation of apocynin-sensitive NADPH oxidase (Nox2) activity in INS-1 832/13 cells under glucotoxic conditions.  

PubMed

Several lines of recent evidence provided compelling evidence to suggest increased generation of reactive oxygen species (ROS) as causal for mitochondrial dysregulation and apoptosis in islet ?-cells exposed to noxious stimuli including high glucose, lipids and proinflammatory cytokines. Studies along these lines are also suggestive of a significant contributory role for NADPH oxidase in the generation of ROS under the above conditions. We have recently reported a marked increase in the expression and activation of cytosolic components of NADPH oxidase (p47phox, Rac1) in cell culture models of glucotoxicity and in islets from T2DM animals (Zucker Diabetic Fatty rat) and humans. In this communication, we provide further evidence indicating significant activation of NADPH activity (~2-fold) in INS-1 832/13 cells exposed to chronic hyperglycemic conditions (20 mM; 48 h). We also report marked attenuation of this activity, by apocynin, a selective inhibitor of phagocyte-like NADPH oxidase (Nox2) activity. Together, our findings implicate Nox2 as a source for ROS generation in ?-cells exposed to glucotoxic conditions. PMID:23695780

Mohammed, Abiy M; Kowluru, Anjaneyulu

2013-05-21

36

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.

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

2012-01-01

37

NADH\\/NADPH Oxidase and Vascular Function  

Microsoft Academic Search

The vascular NADH\\/NADPH oxidase has been shown to be the major source of superoxide in the vessel wall. Recent work has provided insight into its structure and activity in vascular cells. This enzyme is involved in both vascular smooth muscle hypertrophy and in some forms of impaired endothelium-dependent relaxation. Because oxidative stress in general participates in the pathogenesis of hypertension

Kathy K Griendling; Masuko Ushio-Fukai

1997-01-01

38

The Macrolide Roxithromycin Impairs NADPH Oxidase Activation and Alters Translocation of Its Cytosolic Components to the Neutrophil Membrane In Vitro  

Microsoft Academic Search

We have studied the interference of roxithromycin with NADPH oxidase, the key enzymatic system for oxidant production by human neutrophils. Roxithromycin alters the reconstitution of an active enzyme and impairs the translocation to the outer membrane of the cytosolic components p47-phox and p67-phox. Inter- estingly, in resting cells roxithromycin directly triggers the translocation of these factors without stimulating the oxidative

Houria Abdelghaffar; Catherine Babin-Chevaye; Marie-Therese Labro

2005-01-01

39

NADPH oxidases and cardiac remodelling  

Microsoft Academic Search

A heart under chronic stress undergoes cardiac remodelling, a process that comprises structural and functional changes including\\u000a cardiomyocyte hypertrophy, interstitial fibrosis, contractile dysfunction, cell death and ventricular dilatation. Reactive\\u000a oxygen species (ROS)-dependent modulation of intracellular signalling is implicated in the development of cardiac remodelling.\\u000a Among the different ROS sources that are present in the heart, NADPH oxidases (NOXs) are particularly

Adam Nabeebaccus; Min Zhang; Ajay M. Shah

2011-01-01

40

Serotonin 2A and 2B receptor-induced phrenic motor facilitation: differential requirement for spinal NADPH oxidase activity  

PubMed Central

Acute intermittent hypoxia (AIH) facilitates phrenic motor output by a mechanism that requires spinal serotonin (type 2) receptor activation, NADPH oxidase activity and formation of reactive oxygen species (ROS). Episodic spinal serotonin (5-HT) receptor activation alone, without changes in oxygenation, is sufficient to elicit NADPH oxidase-dependent phrenic motor facilitation (pMF). Here we investigated: 1) whether serotonin 2A and/or 2B (5-HT2a/b) receptors are expressed in identified phrenic motor neurons, and 2) which receptor subtype is capable of eliciting NADPH-oxidase-dependent pMF. In anesthetized, artificially ventilated adult rats, episodic C4 intrathecal injections (3 × 6µl injections, 5 min intervals) of a 5-HT2a (DOI) or 5-HT2b (BW723C86) receptor agonist elicited progressive and sustained increases in integrated phrenic nerve burst amplitude (i.e. pMF), an effect lasting at least 90 minutes post-injection for both receptor subtypes. 5-HT2a and 5-HT2b receptor agonist-induced pMF were both blocked by selective antagonists (ketanserin and SB206553, respectively), but not by antagonists to the other receptor subtype. Single injections of either agonist failed to elicit pMF, demonstrating a need for episodic receptor activation. Phrenic motor neurons retrogradely labeled with cholera toxin B fragment expressed both 5-HT2a and 5-HT2b receptors. Pre-treatment with NADPH oxidase inhibitors (apocynin and DPI) blocked 5-HT2b, but not 5-HT2a-induced pMF. Thus, multiple spinal type 2 serotonin receptors elicit pMF, but they act via distinct mechanisms that differ in their requirement for NADPH oxidase activity.

MacFarlane, P.M.; Vinit, S.; Mitchell, G.S.

2011-01-01

41

Ceramide kinase regulates TNF?-stimulated NADPH oxidase activity and eicosanoid biosynthesis in neuroblastoma cells.  

PubMed

A persistent inflammatory reaction is a hallmark of chronic and acute pathologies in the central nervous system (CNS) and greatly exacerbates neuronal degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNF?) plays a pivotal role in the initiation and progression of inflammatory processes provoking oxidative stress, eicosanoid biosynthesis, and the production of bioactive lipids. We established in neuronal cells that TNF? exposure dramatically increased Mg(2+)-dependent neutral sphingomyelinase (nSMase) activity thus generating the bioactive lipid mediator ceramide essential for subsequent NADPH oxidase (NOX) activation and oxidative stress. Since many of the pleiotropic effects of ceramide are attributable to its metabolites, we examined whether ceramide kinase (CerK), converting ceramide to ceramide-1-phosphate, is implicated both in NOX activation and enhanced eicosanoid production in neuronal cells. In the present study, we demonstrated that TNF? exposure of human SH-SY5Y neuroblastoma caused a profound increase in CerK activity. Depleting CerK activity using either siRNA or pharmacology completely negated NOX activation and eicosanoid biosynthesis yet, more importantly, rescued neuronal viability in the presence of TNF?. These findings provided evidence for a critical function of ceramide-1-phospate and thus CerK activity in directly linking sphingolipid metabolism to oxidative stress. This vital role of CerK in CNS inflammation could provide a novel therapeutic approach to intervene with the adverse consequences of a progressive CNS inflammation. PMID:22230689

Barth, Brian M; Gustafson, Sally J; Hankins, Jody L; Kaiser, James M; Haakenson, Jeremy K; Kester, Mark; Kuhn, Thomas B

2011-12-30

42

p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase.  

PubMed

The accumulation of p-cresyl sulfate (PCS), a uremic toxin, is associated with the mortality rate of chronic kidney disease patients; however, the biological functions and the mechanism of its action remain largely unknown. Here we determine whether PCS enhances the production of reactive oxygen species (ROS) in renal tubular cells resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human tubular epithelial cells by enhancing NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activity. PCS also upregulated mRNA levels of inflammatory cytokines and active TGF-?1 protein secretion associated with renal fibrosis. Knockdown of p22(phox) or Nox4 expression suppressed the effect of PCS, underlining the importance of NADPH oxidase activation on its mechanism of action. PCS also reduced cell viability by increasing ROS production. The toxicity of PCS was largely suppressed in the presence of probenecid, an organic acid transport inhibitor. Administration of PCS for 4 weeks caused significant renal tubular damage in 5/6-nephrectomized rats by enhancing oxidative stress. Thus, the renal toxicity of PCS is attributed to its intracellular accumulation, leading to both increased NADPH oxidase activity and ROS production, which, in turn, triggers induction of inflammatory cytokines involved in renal fibrosis. This mechanism is similar to that for the renal toxicity of indoxyl sulfate. PMID:23325087

Watanabe, Hiroshi; Miyamoto, Yohei; Honda, Daisuke; Tanaka, Hisae; Wu, Qiong; Endo, Masayuki; Noguchi, Tsuyoshi; Kadowaki, Daisuke; Ishima, Yu; Kotani, Shunsuke; Nakajima, Makoto; Kataoka, Keiichiro; Kim-Mitsuyama, Shokei; Tanaka, Motoko; Fukagawa, Masafumi; Otagiri, Masaki; Maruyama, Toru

2013-01-16

43

Human Urotensin II Is a Novel Activator of NADPH Oxidase in Human Pulmonary Artery Smooth Muscle Cells  

Microsoft Academic Search

Background—Human urotensin II (hU-II) is a potent vasoactive peptide possibly involved in pulmonary hypertension. Because the signaling mechanisms activated by this peptide in the pulmonary vasculature are largely unknown, we investigated the role of hU-II in the activation of NADPH oxidase and the control of redox-sensitive kinase pathways, expression of plasminogen activator inhibitor-1 (PAI-1), and proliferation in pulmonary artery smooth

Talija Djordjevic; Rachida S. BelAiba; Steve Bonello; Josef Pfeilschifter; John Hess; Agnes Gorlach

2010-01-01

44

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; Chatilialoglu, Alexandros; Steinberg, Benjamin E; Ueyama, Takehiko; Du, Guangwei; Grinstein, Sergio; Fairn, Gregory D

2013-06-27

45

Mechanical strain-induced RhoA activation requires NADPH oxidase-mediated ROS generation in caveolae.  

PubMed

Increased intraglomerular pressure leads to kidney fibrosis, and can be modeled by exposing glomerular mesangial cells (MC) to mechanical strain. We previously showed that RhoA mediates strain-induced matrix production. Here we investigate whether reactive oxygen species (ROS) are required for RhoA activation. Maximal RhoA activation (1 min) was inhibited by ROS scavenge or NADPH oxidase inhibition. Strain activated NADPH oxidase, with Rac1, p47(phox), and p67(phox) membrane translocation, and Rac1 activation, observed within 30 sec. Epidermal growth factor receptor (EGFR) inhibition blocked RhoA and Rac1 activation, p67(phox) membrane translocation, and ROS generation. However, EGFR activation was unaffected by ROS inhibitors, placing it upstream of ROS generation. We previously showed, using chemical disruption, that caveolae mediate strain-induced EGFR and RhoA activation. In MC from caveolin-1 knockout mice, which lack caveolae, RhoA and Rac1 activation, p67(phox) membrane translocation, and ROS generation were absent. These were rescued by caveolin-1 re-expression. ROS generation, Rac1 activation, and p67(phox) membrane translocation were also prevented by Src inhibition. They were absent in MC stably infected with caveolin-1 Y14A, a mutant resistant to Src phosphorylation. In MC, caveolae are thus important mediators of strain-induced ROS generation through NADPH oxidase, mediating a signaling cascade which results in RhoA activation. PMID:20380579

Zhang, Ying; Peng, Fangfang; Gao, Bo; Ingram, Alistair J; Krepinsky, Joan C

2010-10-01

46

NMDA receptor-mediated activation of NADPH oxidase and glomerulosclerosis in hyperhomocysteinemic rats.  

PubMed

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 O(2)(.-) production in the glomeruli, which were accompanied by remarkable glomerulosclerosis. MK-801 treatment significantly inhibited Nox-dependent O(2)(.-) 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 O(2)(.-) 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. PMID:20406136

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

2010-10-01

47

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.

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

2010-01-01

48

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

PubMed Central

Background Recent 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 NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat. Methodology/Principal Findings Our studies revealed that NADPH oxidase activity and superoxide (O2?) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O2? production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated “in situ” O2? production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7–9 d post reperfusion as compared to vehicle-treated animals. Conclusions/Significance The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.

Raz, Limor; Zhang, Quan-Guang; Zhou, Cai-feng; Han, Dong; Gulati, Priya; Yang, Li-cai; Yang, Fang; Wang, Rui-min; Brann, Darrell W.

2010-01-01

49

NADPH oxidase mediated oxidative stress in hepatic fibrogenesis  

PubMed Central

NADPH oxidase (NOX) is a multicomponent enzyme complex that generates reactive oxygen species (ROS) in response to a wide range of stimuli. ROS is involved as key secondary messengers in numerous signaling pathways, and NADPH oxidases complex has been increasingly recognized as key elements of intracellular signaling of hepatic fibrogenesis. In the liver, NADPH oxidase is functionally expressed both in the phagocytic form and in the non-phagocytic form. The non-phagocytic NADPH oxidase complex is structurally and functionally similar to the phagocytic NADPH, resulting in reduction of molecular oxygen to generate superoxide. There are six homologous NOX proteins in the non-phagocytic forms of NADPH oxidase. An emerging concept is that both phagocytic and nonphagocytic NADPH oxidase components in hepatic stellate cells (HSCs) mediate hepatic fibrosis, suggesting its potential role as a pharmacological target for anti-fibrotic therapy. The molecular components and signaling pathways of various NADPH oxidase homologues that are critical for the fibrotic activity in HSCs need to be more clearly identified.

Brenner, David A.

2011-01-01

50

Contribution of NADPH oxidase to membrane CD38 internalization and activation in coronary arterial myocytes.  

PubMed

The CD38-ADP-ribosylcyclase-mediated Ca(2+) signaling pathway importantly contributes to the vasomotor response in different arteries. Although there is evidence indicating that the activation of CD38-ADP-ribosylcyclase is associated with CD38 internalization, the molecular mechanism mediating CD38 internalization and consequent activation in response to a variety of physiological and pathological stimuli remains poorly understood. Recent studies have shown that CD38 may sense redox signals and is thereby activated to produce cellular response and that the NADPH oxidase isoform, NOX1, is a major resource to produce superoxide (O2 (·-) ) in coronary arterial myocytes (CAMs) in response to muscarinic receptor agonist, which uses CD38-ADP-ribosylcyclase signaling pathway to exert its action in these CAMs. These findings led us hypothesize that NOX1-derived O2 (·-) serves in an autocrine fashion to enhance CD38 internalization, leading to redox activation of CD38-ADP-ribosylcyclase activity in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2 (·-) production and increased CD38-ADP-ribosylcyclase activity in CAMs, while activation of NOX1 by overexpression of Rac1 or Vav2 or administration of exogenous O2 (·-) significantly increased CD38 internalization in CAMs. Lastly, ET-1 was found to markedly increase membrane raft clustering as shown by increased colocalization of cholera toxin-B with CD38 and NOX1. Taken together, these results provide direct evidence that Rac1-NOX1-dependent O2 (·-) production mediates CD38 internalization in CAMs, which may represent an important mechanism linking receptor activation with CD38 activity in these cells. PMID:23940720

Xu, Ming; Li, Xiao-Xue; Ritter, Joseph K; Abais, Justine M; Zhang, Yang; Li, Pin-Lan

2013-08-07

51

Mutations in the PX-SH3A Linker of p47phox Decouple PI(3,4)P2 Binding from NADPH Oxidase Activation  

PubMed Central

NADPH oxidase is essential in the human innate immune response. p47phox, a cytosolic NADPH oxidase component, plays a regulatory role in the activation of NADPH oxidase. Our manipulation of p47phox by mutation and amino acid deletion shows that the linker region between the PX and N-terminal SH3 domain plays a role in blocking the binding of the phosphoinositide 3,4-bisphosphate [PI(3,4)P2], a lipid second messenger generated upon neutrophil activation. Replacement of linker residues 151–158 with glycine alters NMR-measured spin lattice relaxation rates and sedimentation velocity compared to those of the wild-type protein, suggesting that the PX domain is released from its autoinhibited conformation. Liposome binding and surface plasmon resonance experiments confirm this result, showing that this mutant has a similar binding affinity for the isolated PX domain toward PI(3,4)P2. However, an in vitro NADPH oxidase activity assay reveals that this glycine mutant of the full-length protein greatly reduced NADPH oxidase activity upon activation even though it displayed PI(3,4)P2 binding activity comparable to that of the isolated PX domain. Our results highlight an active role of the PX–SH3 linker region in maintaining p47phox in its fully autoinhibited form and demonstrate that binding of p47phox to membrane phospholipids is mechanistically distinct from NADPH oxidase activation.

Shen, Kai; Sergeant, Susan; Hantgan, Roy R.; McPhail, Linda C.; Horita, David A.

2009-01-01

52

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

PubMed

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 HD(140Q/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 HD(140Q/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 HD(140Q/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 HD(140Q/140Q) neurons. HD(140Q/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 HD(140Q/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

2012-12-07

53

Protein kinase C-dependent NAD(P)H oxidase activation induced by type 1 diabetes in renal medullary thick ascending limb.  

PubMed

Type 1 diabetes provokes a protein kinase C (PKC)-dependent accumulation of superoxide anion in the renal medullary thick ascending limb (mTAL). We hypothesized that this phenomenon involves PKC-dependent NAD(P)H oxidase activation. The validity of this hypothesis was explored using mTAL suspensions prepared from rats with streptozotocin-induced diabetes and from sham (vehicle-treated) rats. Superoxide production was 5-fold higher in mTAL suspensions from diabetic rats compared with suspensions from sham rats. The NAD(P)H oxidase inhibitor apocynin caused an 80% decrease in superoxide production by mTAL from diabetic rats (P<0.05 vs untreated) without altering superoxide production by sham mTAL. NAD(P)H oxidase activity was >2-fold higher in mTAL from diabetic rats than in sham mTAL (P<0.05). Pretreatment with calphostin C (broad-spectrum PKC inhibitor) or rottlerin (PKCdelta inhibitor) reduced NAD(P)H oxidase activity by approximately 80% in both groups; however, PKCalpha/beta or PKCbeta inhibition did not alter NAD(P)H oxidase activity in either group. Protein levels of Nox2, Nox4, and p47phox were significantly higher in diabetic mTAL than in mTAL from sham rats. In summary, elevated superoxide production by mTAL from diabetic rats was normalized by NAD(P)H oxidase inhibition. PKC-dependent, PKCdelta-dependent, and total NAD(P)H oxidase activity was greater in mTAL from diabetic rats compared with sham. Protein levels of Nox2, Nox4, and p47phox were increased in mTAL from diabetic rats. We conclude that increased superoxide production by the mTAL during diabetes involves a PKCdelta-dependent increase in NAD(P)H oxidase activity in concert with increased protein levels of catalytic and regulatory subunits of the enzyme. PMID:20038746

Yang, Jing; Lane, Pascale H; Pollock, Jennifer S; Carmines, Pamela K

2009-12-28

54

Prolonged exposure to LPS increases iron, heme, and p22phox levels and NADPH oxidase activity in human aortic endothelial cells: Inhibition by desferrioxamine  

PubMed Central

Objective Vascular oxidative stress and inflammation are contributing factors in atherosclerosis. We recently found that the iron chelator, desferrioxamine (DFO), suppresses NADPH oxidase-mediated oxidative stress and expression of cellular adhesion molecules in mice treated with lipopolysaccharide (LPS). The objective of the present study was to investigate whether and how LPS and iron enhance, and DFO inhibits, NADPH oxidase activity in human aortic endothelial cells (HAEC). Methods and Results Incubation of HAEC for 24 hrs with 5 ?g/mL LPS led to a four-fold increase in NADPH oxidase activity, which was strongly suppressed by pretreatment of the cells for 24 hrs with 100 ?mol/L DFO. Incubating HAEC with LPS also significantly increased cellular iron and heme levels and mRNA and protein levels of p22phox, a heme-containing, catalytic subunit of NADPH oxidase. All of these effects of LPS on HAEC were strongly inhibited by DFO. Exposing HAEC to 100 mol/L iron (ferric citrate) for 48 hrs exerted similar effects as LPS, and these effects were strongly inhibited by co-incubation with DFO. Furthermore, neither LPS nor DFO affected mRNA and protein levels of p47phox, a non-heme containing, regulatory subunit of NADPH oxidase, or the mRNA level of NOX4, an isoform of the principal catalytic subunit of NADPH oxidase in endothelial cells. In contrast, heme oxygenase-1 was strongly suppressed by DFO, both in the absence and presence of LPS or iron. Conclusions Our data indicate that prolonged exposure to LPS or iron increases endothelial NADPH oxidase activity by increasing p22phox gene transcription and cellular levels of iron, heme, and p22phox protein. Iron chelation by DFO effectively suppresses endothelial NADPH oxidase activity, which may be helpful as an adjunct in reducing vascular oxidative stress and inflammation in atherosclerosis.

Li, Lixin; Frei, Balz

2009-01-01

55

Rotenone Activates Phagocyte NADPH Oxidase through Binding to Its Membrane Subunit gp91phox  

PubMed Central

Rotenone, a widely used pesticide, reproduces Parkinsonism in rodents and associates with increased risk for Parkinson’s disease. We previously reported rotenone increased superoxide production through stimulating microglial phagocyte NADPH oxidase (PHOX). The present study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91phox, the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91phox. Functional studies showed both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91phox/p22phox) and cytosolic subunits (p67phox and p47phox). Rotenone-elicited extracellular superoxide release in p47phox-deficient macrophages suggested rotenone enabled to activate PHOX through a p47phox-independent mechanism. Increased membrane translocation of p67phox, elevated binding of p67phox to rotenone-treated membrane fractions, and co-immunoprecipitation of p67phox and gp91phox in rotenone-treated wild-type and p47phox-deficient macrophages indicated p67phox played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91phox. Rac1, a Rho-like small GTPase, enhanced p67phox-gp91phox interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91phox; such an interaction triggered membrane translocation of p67phox, leading to PHOX activation and superoxide production.

Zhou, Hui; Zhang, Feng; Chen, Shih-heng; Zhang, Dan; Wilson, Belinda; Hong, Jau-shyong; Gao, Hui-Ming

2011-01-01

56

Epithelial-to-mesenchymal transition in podocytes mediated by activation of NADPH oxidase in hyperhomocysteinemia.  

PubMed

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 (O??) 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 gp91( phox ) (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 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

2011-06-07

57

Comparative pharmacology of chemically distinct NADPH oxidase inhibitors  

PubMed Central

BACKGROUND AND PURPOSE Oxidative stress [i.e. increased levels of reactive oxygen species (ROS)] has been suggested as a pathomechanism of different diseases, although the disease-relevant sources of ROS remain to be identified. One of these sources may be NADPH oxidases. However, due to increasing concerns about the specificity of the compounds commonly used as NADPH oxidase inhibitors, data obtained with these compounds may have to be re-interpreted. EXPERIMENTAL APPROACH We compared the pharmacological profiles of the commonly used NADPH oxidase inhibitors, diphenylene iodonium (DPI), apocynin and 4-(2-amino-ethyl)-benzolsulphonyl-fluoride (AEBSF), as well as the novel triazolo pyrimidine VAS3947. We used several assays for detecting cellular and tissue ROS, as none of them is specific and artefact free. KEY RESULTS DPI abolished NADPH oxidase-mediated ROS formation, but also inhibited other flavo-enzymes such as NO synthase (NOS) and xanthine oxidase (XOD). Apocynin interfered with ROS detection and varied considerably in efficacy and potency, as did AEBSF. Conversely, the novel NADPH oxidase inhibitor, VAS3947, consistently inhibited NADPH oxidase activity in low micromolar concentrations, and interfered neither with ROS detection nor with XOD or eNOS activities. VAS3947 attenuated ROS formation in aortas of spontaneously hypertensive rats (SHRs), where NOS or XOD inhibitors were without effect. CONCLUSIONS AND IMPLICATIONS Our data suggest that triazolo pyrimidines such as VAS3947 are specific NADPH oxidase inhibitors, while DPI and apocynin can no longer be recommended. Based on the effects of VAS3947, NADPH oxidases appear to be a major source of ROS in aortas of SHRs.

Wind, S; Beuerlein, K; Eucker, T; Muller, H; Scheurer, P; Armitage, ME; Ho, H; Schmidt, HHHW; Wingler, K

2010-01-01

58

Novel isoforms of NADPH-oxidase in cerebral vascular control.  

PubMed

Reactive oxygen species (ROS) are thought to play an important role in the initiation and progression of a variety of vascular diseases. Furthermore, accumulating evidence indicates that ROS may also serve as important cell signalling molecules for the regulation of normal vascular function. Recently, a novel family of proteins (Nox1, 2 and 4) that act as the catalytic subunit of the superoxide (O2-) producing enzyme NADPH-oxidase has been discovered in vascular cells. There is now preliminary evidence suggesting that NADPH-oxidase-derived ROS may serve as a physiological vasodilator mechanism in the cerebral circulation. Moreover, the activity of NADPH-oxidase is profoundly greater in cerebral versus systemic arteries. Studies have shown that Nox1, Nox2 (also known as gp91phox) and Nox4 are all expressed in cerebral arteries, suggesting that multiple isoforms of NADPH-oxidase may be important for ROS production by cerebral arteries. Enhanced NADPH-oxidase activity is associated with several vascular-related diseases, including hypertension, stroke, subarachnoid haemorrhage and Alzheimer's dementia; however, the consequences of this for cerebral vascular function are controversial. For example, there is some evidence suggesting that NADPH-oxidase-derived O2- may play a role in endothelial dysfunction of cerebral arteries and a subsequent rise in cerebral vascular tone, associated with hypertension. However, activation of NADPH-oxidase elicits cerebral vasodilatation in vivo, and this mechanism is enhanced in chronic hypertension. While further supportive evidence is needed, it is an intriguing possibility that NADPH-oxidase-derived ROS may play a protective role in regulating cerebral vascular tone during disease. PMID:16616784

Miller, Alyson A; Drummond, Grant R; Sobey, Christopher G

2006-04-17

59

D1-like receptors regulate NADPH oxidase activity and subunit expression in lipid raft microdomains of renal proximal tubule cells.  

PubMed

NADPH oxidase (Nox)-dependent reactive oxygen species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than in normotensive rats. However, more Rac1 was expressed in lipid rafts in cells from hypertensive rats than in cells from normotensive rats; the converse was observed with Nox4, whereas Nox2 expression was similar. The D(1)-like receptor agonist fenoldopam decreased Nox2 and Rac1 protein in lipid rafts to a greater extent in hypertensive than in normotensive rats. Basal oxidase activity was 3-fold higher in hypertensive than in normotensive rats but was inhibited to a greater extent by fenoldopam in normotensive (58+/-3.3%) than in hypertensive rats (31+/-5.2%; P<0.05; n=6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67(phox) in cells from normotensive rats. D(1)-like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl-beta-cyclodextrin decreased oxidase activity and cholesterol content to a greater extent in hypertensive than in normotensive rats. The greater basal levels of Nox2 and Nox4 in cell membranes and Nox2 and Rac1 in lipid rafts in hypertensive rats than in normotensive rats may explain the increased basal oxidase activity in hypertensive rats. PMID:19380616

Li, Hewang; Han, Weixing; Villar, Van Anthony M; Keever, Lindsay B; Lu, Quansheng; Hopfer, Ulrich; Quinn, Mark T; Felder, Robin A; Jose, Pedro A; Yu, Peiying

2009-04-20

60

ADAM17 mediates Nox4 expression and NADPH oxidase activity in the kidney cortex of OVE26 mice.  

PubMed

Matrix protein accumulation is a prominent feature of diabetic nephropathy that contributes to renal fibrosis and decline in renal function. The pathogenic mechanisms of matrix accumulation are incompletely characterized. We investigated if the matrix metalloprotease a disintegrin and metalloprotease1 7 (ADAM17), known to cleave growth factors and cytokines, is activated in the kidney cortex of OVE26 type 1 diabetic mice and the potential mechanisms by which ADAM17 mediates extracellular matrix accumulation. Protein expression and activity of ADAM17 were increased in OVE26 kidney cortex. Using a pharmacological inhibitor to ADAM17, TMI-005, we determined that ADAM17 activation results in increased type IV collagen, Nox4, and NADPH oxidase activity in the kidney cortex of diabetic mice. In cultured mouse proximal tubular epithelial cells (MCTs), high glucose increases ADAM17 activity, Nox4 and fibronectin expression, cellular collagen content, and NADPH oxidase activity. These effects of glucose were inhibited when cells were pretreated with TMI-005 and/or transfected with small interfering ADAM17. Collectively, these data indicate a novel mechanism whereby hyperglycemia in diabetes increases extracellular matrix protein expression in the kidney cortex through activation of ADAM17 and enhanced oxidative stress through Nox enzyme activation. Additionally, our study is the first to provide evidence that Nox4 is downstream of ADAM17. PMID:23678045

Ford, Bridget M; Eid, Assaad A; Gö?z, Monika; Barnes, Jeffrey L; Gorin, Yves C; Abboud, Hanna E

2013-05-15

61

A transverse tubule NADPH oxidase activity stimulates calcium release from isolated triads via ryanodine receptor type 1 S -glutathionylation.  

PubMed

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91(phox) membrane subunit and the cytoplasmic regulatory p47(phox) subunit co-localized in transverse tubules of adult mice fibers with the alpha1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91(phox) and p22(phox), which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47(phox) and p67(phox). NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed. PMID:16762927

Hidalgo, Cecilia; Sánchez, Gina; Barrientos, Genaro; Aracena-Parks, Paula

2006-06-08

62

Inhibition of NADPH Oxidase by Apocynin Attenuates Progression of Atherosclerosis  

PubMed Central

Of the multiple sources of reactive oxygen species (ROS) in the blood vessel, NADPH oxidases are the primary source. Whereas several studies have implicated NADPH oxidases in the initiation of atherosclerosis, their roles in disease progression are incompletely understood. Our objective was to determine the potential clinical relevance of inhibiting NADPH oxidase in established atherosclerosis. Using a hypercholesteremic murine model of atherosclerosis (ApoE?/?/LDLR?/? (AS) mice on normal chow diet), we first established a time-dependent relationship between superoxide levels and lesion size in AS mice. Next, we identified NADPH oxidase as the primary source of ROS in atherosclerotic lesions. Treatment of aortic segments from AS mice with apocynin, which interferes with NADPH oxidase activation in part by preventing translocation of the subunit p47phox, significantly reduced superoxide levels. Moreover, addition of apocynin to the drinking water of AS mice produced a decrease in lesion size as compared to untreated AS mice, with the effect most pronounced in the thoracoabdominal aorta but absent from the aortic arch. Granulocyte function in AS+apocynin mice was suppressed, confirming efficacy of apocynin treatment. We conclude that apocynin attenuates the progression of atherosclerosis in hypercholesterolemic mice, potentially by its ability to inhibit generation of superoxide by NADPH oxidase.

Kinkade, Kara; Streeter, Jennifer; Miller, Francis J.

2013-01-01

63

A nonpolar blueberry fraction blunts NADPH oxidase activation in neuronal cells exposed to tumor necrosis factor-?.  

PubMed

Inflammation and oxidative stress are key to the progressive neuronal degeneration common to chronic pathologies, traumatic injuries, and aging processes in the CNS. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-?) orchestrates cellular stress by stimulating the production and release of neurotoxic mediators including reactive oxygen species (ROS). NADPH oxidases (NOX), ubiquitously expressed in all cells, have recently emerged as pivotal ROS sources in aging and disease. We demonstrated the presence of potent NOX inhibitors in wild Alaska bog blueberries partitioning discretely into a nonpolar fraction with minimal antioxidant capacity and largely devoid of polyphenols. Incubation of SH-SY5Y human neuroblastoma cells with nonpolar blueberry fractions obstructed the coalescing of lipid rafts into large domains disrupting NOX assembly therein and abolishing ROS production characteristic for TNF-? exposure. These findings illuminate nutrition-derived lipid raft modulation as a novel therapeutic approach to blunt inflammatory and oxidative stress in the aging or diseased CNS. PMID:22530077

Gustafson, Sally J; Dunlap, Kriya L; McGill, Colin M; Kuhn, Thomas B

2012-03-13

64

A Nonpolar Blueberry Fraction Blunts NADPH Oxidase Activation in Neuronal Cells Exposed to Tumor Necrosis Factor-?  

PubMed Central

Inflammation and oxidative stress are key to the progressive neuronal degeneration common to chronic pathologies, traumatic injuries, and aging processes in the CNS. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-?) orchestrates cellular stress by stimulating the production and release of neurotoxic mediators including reactive oxygen species (ROS). NADPH oxidases (NOX), ubiquitously expressed in all cells, have recently emerged as pivotal ROS sources in aging and disease. We demonstrated the presence of potent NOX inhibitors in wild Alaska bog blueberries partitioning discretely into a nonpolar fraction with minimal antioxidant capacity and largely devoid of polyphenols. Incubation of SH-SY5Y human neuroblastoma cells with nonpolar blueberry fractions obstructed the coalescing of lipid rafts into large domains disrupting NOX assembly therein and abolishing ROS production characteristic for TNF-? exposure. These findings illuminate nutrition-derived lipid raft modulation as a novel therapeutic approach to blunt inflammatory and oxidative stress in the aging or diseased CNS.

Gustafson, Sally J.; Dunlap, Kriya L.; McGill, Colin M.; Kuhn, Thomas B.

2012-01-01

65

Activation of p38 MAPK induced peroxynitrite generation in LPS plus IFN-gamma-stimulated rat primary astrocytes via activation of iNOS and NADPH oxidase.  

PubMed

We have shown that immunostimulated astrocytes produce excess nitric oxide (NO) and eventually peroxynitrite (ONOO(-)) that was closely associated with the glucose deprivation-potentiated death of astrocytes. The present study shows that activated p38 MAPK regulates ONOO(-) generation from lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-stimulated astrocytes. LPS+IFN-gamma-induced p38 MAPK activation and ONOO(-) generation were attenuated by SB203580 or SKF-86002, specific inhibitors of p38 MAPK. ONOO(-) generation was blocked by NADPH oxidase inhibitor, diphenyleneiodonium chloride, and nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester, suggesting both enzymes are involved in ONOO(-) generation. Inhibition of p38 MAPK suppressed LPS+IFN-gamma-induced NO production through down-regulating inducible form of NOS expression. It also suppressed LPS+IFN-gamma-induced NADPH oxidase activation and eventually, the inducible form of superoxide production. Transfection with dominant negative vector of p38 alpha reduced LPS+IFN-gamma-induced ONOO(-) generation through blocking both iNOS-derived NO production and NADPH oxidase-derived O2(-) production. Our results suggest that activated p38 MAPK may serve as a potential signaling molecule in ONOO(-) generation through dual regulatory mechanisms, involving iNOS induction and NADPH oxidase activation. PMID:18289732

Yoo, Byoung Kwon; Choi, Ji Woong; Shin, Chan Young; Jeon, Se Jin; Park, Seo Jin; Cheong, Jae Hoon; Han, Sun Young; Ryu, Jae Ryun; Song, Mi Ryoung; Ko, Kwang Ho

2007-12-27

66

Sustained activation of proton channels and NADPH oxidase in human eosinophils and murine granulocytes requires PKC but not cPLA2? activity  

PubMed Central

The prevailing hypothesis that a signalling pathway involving cPLA2? is required to enhance the gating of the voltage-gated proton channel associated with NADPH oxidase was tested in human eosinophils and murine granulocytes. This hypothesis invokes arachidonic acid (AA) liberated by cPLA2? as a final activator of proton channels. In human eosinophils studied in the perforated-patch configuration, phorbol myristate acetate (PMA) stimulation elicited NADPH oxidase-generated electron current (Ie) and enhanced proton channel gating identically in the presence or absence of three specific cPLA2? inhibitors, Wyeth-1, pyrrolidine-2 and AACOCF3 (arachidonyl trifluoromethyl ketone). In contrast, PKC inhibitors GFX (GF109203X) or staurosporine prevented the activation of either proton channels or NADPH oxidase. PKC inhibition during the respiratory burst reversed the activation of both molecules, suggesting that ongoing phosphorylation is required. This effect of GFX was inhibited by okadaic acid, implicating phosphatases in proton channel deactivation. Proton channel activation by AA was partially reversed by GFX or staurosporine, indicating that AA effects are due in part to activation of PKC. In granulocytes from mice with the cPLA2? gene disrupted (knockout mice), PMA or fMetLeuPhe activated NADPH oxidase and proton channels in a manner indistinguishable from the responses of control cells. Thus, cPLA2? is not essential to activate the proton conductance or for a normal respiratory burst. Instead, phosphorylation of the proton channel or an activating molecule converts the channel to its activated gating mode. The existing paradigm for regulation of the concerted activity of proton channels and NADPH oxidase must be revised.

Morgan, Deri; Cherny, Vladimir V; Finnegan, Alison; Bollinger, James; Gelb, Michael H; DeCoursey, Thomas E

2007-01-01

67

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

PubMed

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; Helms, My N

2013-07-17

68

Oxalate-induced activation of PKC-? and -? regulates NADPH oxidase-mediated oxidative injury in renal tubular epithelial cells  

PubMed Central

Oxalate-induced oxidative stress contributes to cell injury and promotes renal deposition of calcium oxalate crystals. However, we do not know how oxalate stimulates reactive oxygen species (ROS) in renal tubular epithelial cells. We investigated the signaling mechanism of oxalate-induced ROS formation in these cells and found that oxalate significantly increased membrane-associated protein kinase C (PKC) activity while at the same time lowering cytosolic PKC activity. Oxalate markedly translocated PKC-? and -? from the cytosol to the cell membrane. Pretreatment of LLC-PK1 cells with specific inhibitors of PKC-? or -? significantly blocked oxalate-induced generation of superoxide and hydrogen peroxide along with NADPH oxidase activity, LDH release, lipid hydroperoxide formation, and apoptosis. The PKC activator PMA mimicked oxalate's effect on oxidative stress in LLC-PK1 cells as well as cytosol-to-membrane translocation of PKC-? and -?. Silencing of PKC-? expression by PKC-?-specific small interfering RNA significantly attenuated oxalate-induced cell injury by decreasing hydrogen peroxide generation and LDH release. We believe this is the first demonstration that PKC-?- and -?-dependent activation of NADPH oxidase is one of the mechanisms responsible for oxalate-induced oxidative injury in renal tubular epithelial cells. The study suggests that the therapeutic approach might be considered toward attenuating oxalate-induced PKC signaling-mediated oxidative injury in recurrent stone formers.

Menon, Mani; Thamilselvan, Sivagnanam

2009-01-01

69

Kaempferol modulates pro-inflammatory NF-?B activation by suppressing advanced glycation endproducts-induced NADPH oxidase  

PubMed Central

Advanced glycation endproducts (AGE) are oxidative products formed from the reaction between carbohydrates and a free amino group of proteins that are provoked by reactive species (RS). It is also known that AGE enhance the generation of RS and that the binding of AGE to a specific AGE receptor (RAGE) induces the activation of the redox-sensitive, pro-inflammatory transcription factor, nuclear factor-kappa B (NF-?B). In this current study, we investigated the anti-oxidative effects of short-term kaempferol supplementation on the age-related formation of AGE and the binding activity of RAGE in aged rat kidney. We further investigated the suppressive action of kaempferol against AGE's ability to stimulate activation of pro-inflammatory NF-?B and its molecular mechanisms. For this study, we utilized young (6 months old), old (24 months old), and kaempferol-fed (2 and 4 mg/kg/day for 10 days) old rats. In addition, for the molecular work, the rat endothelial cell line, YPEN-1 was used. The results show that AGE and RAGE were increased during aging and that these increases were blunted by kaempferol. In addition, dietary kaempferol reduced age-related increases in NF-?B activity and NF-?B-dependant pro-inflammatory gene activity. The most significant new finding from this study is that kaempferol supplementation prevented age-related NF-?B activation by suppressing AGE-induced nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). Taken together, our results demonstrated that dietary kaempferol exerts its anti-oxidative and anti-inflammatory actions by modulating the age-related NF-?B signaling cascade and its pro-inflammatory genes by suppressing AGE-induced NADPH oxidase activation. Based on these data, dietary kaempferol is proposed as a possible anti-AGE agent that may have the potential for use in anti-inflammation therapies.

Kim, Ji Min; Lee, Eun Kyeong; Kim, Dae Hyun; Yu, Byung Pal

2010-01-01

70

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.

Brechard, Sabrina; Plancon, Sebastien

2013-01-01

71

Interleukin-10 Released by CD4+CD25+ Natural Regulatory T Cells Improves Microvascular Endothelial Function Through Inhibition of NADPH Oxidase Activity in Hypertensive Mice  

PubMed Central

Objective We previously demonstrated that a reduced number of CD4+CD25+-regulatory T cells (Tregs) was associated with microvascular dysfunction in hypertension. However, the underlying mechanism by which Tregs regulate vascular endothelial function remains unknown. Methods and Results Control and interleukin (IL)-10–/– knockout mice were infused with angiotensin II (400 ng/kg/min) for 2 weeks (hypertensive [HT] and HT-IL-10–/–). Endothelium-dependent relaxation (EDR) in response to acetylcholine was significantly reduced in mesenteric resistance artery (MRA) from HT and HT-IL-10–/– compared with control and IL-10–/– mice. Importantly, the incubation of MRA from HT mice with the conditioned media of cultured Tregs, isolated from control mice, reduced NADPH oxidase activity and improved EDR, whereas no effect was observed in MRA from control mice incubated with the same media. These effects were reversed when MRAs were preincubated with IL-10 antibody or IL-10 receptor antagonist, whereas incubation with transforming growth factor-? receptor antagonist had no effect. The transfer of cultured Tregs, isolated from control mice, into HT-IL-10–/– mice reduced systolic blood pressure (SBP) and NADPH oxidase activity and improved EDR in MRA compared with untreated HT-IL-10–/– mice. In vivo treatment of HT mice with IL-10 (1000 ng/mouse) significantly reduced SBP and NADPH oxidase activity and improved EDR in MRA compared with untreated HT mice. The transfer of cultured Tregs, isolated from IL-10–/– mice, into HT mice did not reduce SBP or NADPH oxidase activity or improve EDR. The incubation of MRA from HT mice with apocynin improved EDR, whereas NADPH oxidase substrate attenuated EDR in MRA from control mice, which was reversed with exogenous IL-10. Conclusion These data demonstrate that IL-10 released from Tregs attenuates NADPH oxidase activity, which is a critical process in the improvement of microvascular endothelial function in hypertension, suggesting that Tregs/IL-10 could be a therapeutic target for treatment of vasculopathy in hypertension.

Kassan, Modar; Galan, Maria; Partyka, Megan; Trebak, Mohamed; Matrougui, Khalid

2013-01-01

72

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

PubMed

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 Ca(2+) 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

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

2013-01-24

73

Increase in Anthraquinone Content in Rubia cordifolia Cells Transformed by rol Genes Does Not Involve Activation of the NADPH Oxidase Signaling Pathway  

Microsoft Academic Search

It has been reported that rol plant oncogenes located in Ri-plasmids of Agrobacterium rhizogenes activated synthesis of secondary metabolites in the transformed plant cells. The activator mechanism is still unknown. In this work, we studied whether the NADPH oxidase-signaling pathway, which regulates the synthesis of defense metabolites in plants, is involved in the activator function of the rol genes. It

V. P. Bulgakov; G. K. Tchernoded; N. P. Mischenko; Yu. N. Shkryl; V. P. Glazunov; S. A. Fedoreyev; Yu. N. Zhuravlev

2003-01-01

74

P2x7 Receptor-NADPH Oxidase-Axis Mediates Protein radical Formation And Kupffer Cell Activation in Carbon Tetrachloride-Mediated Steatohepatitis in Obese Mice  

PubMed Central

While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl4-treated hepatocytes and generating redoxmediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and post-translational nitration, primarily in Kupffer cells, at 24 h post-CCl4 administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase- and P2X7 receptor-dependent, correlated well with the release of TNF- ? and MCP-2 from Kupffer cells. The Kupffer cells in CCl4-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms.

Chatterjee, Saurabh; Rana, Ritu; Corbett, Jean; Kadiiska, Maria B.; Goldstein, Joyce; Mason, Ronald P.

2012-01-01

75

Neuroprotection conferred by post-ischemia ethanol therapy in experimental stroke: an inhibitory effect on hyperglycolysis and NADPH oxidase activation.  

PubMed

Ethanol provides neuroprotection following ischemia/reperfusion. This study assessed ethanol's effect on hyperglycolysis and NADPH oxidase (NOX) activation. Adult, male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h. Three sets of experiments were conducted to determine ethanol's effect on (i) conferring neuroprotection by measuring infarct volume and neurological deficits 24 h post reperfusion; (ii) cerebral glucose metabolism and lactic acidosis by measuring brain and blood glucose concentrations and protein expression of glucose transporter 1 and 3 (GLUT1, GLUT3), phosphofructokinase (PFK), as well as lactic acidosis by measuring lactate dehydrogenase (LDH), and lactate; and (iii) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activation by detecting enzymatic activity and subunit expression at 3 h after reperfusion. When administered upon reperfusion, ethanol (1.5 g/kg) reduced infarct volume by 40% (p < 0.01) and neurological deficits by 48% at 24 h post reperfusion while reducing (p < 0.01) elevations in glycolytic protein expression and lactate levels during early reperfusion (3 h). Ethanol increased the reductions in cerebral glucose concentration at 3 h post reperfusion by 64% (p < 0.01) while enhancing (p < 0.01) post stroke blood glucose concentration, suggesting a reduced cellular glucose uptake and utilization. Ethanol decreased (p < 0.01) stroke-induced NOX activation by reducing enzymatic activity and gp91(phox) expression by 45% and 38%, respectively. Post-ischemia ethanol treatment exerts neuroprotection through attenuation of hyperglycolysis and associated NOX activation. Because of the lack of associated hypoglycemia and selectivity toward decreasing cerebral metabolism, further investigation of ethanol's use as a post-stroke therapy, especially in the context of hyperglycemia, seems warranted. PMID:23350720

Kochanski, Ryan; Peng, Changya; Higashida, Tetsuhiro; Geng, Xiaokun; Hüttemann, Maik; Guthikonda, Murali; Ding, Yuchuan

2013-02-24

76

Potential role of NADPH oxidase-mediated activation of Jak2\\/Stat3 and mitogen-activated protein kinases and expression of TGF-?1 in the pathophysiology of acute pancreatitis  

Microsoft Academic Search

Objective  NADPH oxidase is potentially associated with acute pancreatitis by producing reactive oxygen species (ROS). We investigated\\u000a whether NADPH oxidase mediates the activation of Janus kinase (Jak)2\\/signal transducers and activators of transcription (Stat)3\\u000a and mitogen-activated protein kinases (MAPKs) to induce the expression of transforming growth factor-?1 (TGF-?1) in cerulein-stimulated\\u000a pancreatic acinar cells.\\u000a \\u000a \\u000a \\u000a \\u000a Treatment  AR42J cells were treated with an NADPH oxidase inhibitor

Kyung Don Ju; Joo Weon Lim; Kyung Hwan Kim; Hyeyoung Kim

77

NADPH OXIDASE IN STROKE AND CEREBROVASCULAR DISEASE  

PubMed Central

NADPH oxidase (NOX) was originally identified in immune cells as playing an important microbicidal role. In stroke and cerebrovascular disease, inflammation is increasingly being recognized as contributing negatively to neurological outcome, with NOX as an important source of superoxide. Several labs have now shown that blocking or deleting NOX in the experimental stroke models protects from brain ischemic. Recent work has implicated glucose as an important NOX substrate leading to reperfusion injury, and that NOX inhibition can improve the detrimental effects of hyperglycemia on stroke. NOX inhibition also appears to ameliorate complications of thrombolytic therapy by reducing blood brain barrier disruption, edema formation and hemorrhage. Further, NOX from circulating inflammatory cells seems to contribute more to ischemic injury more than NOX generated from endogenous brain residential cells. Several pharmacological inhibitors of NOX are now available. Thus, blocking NOX activation may prove to be a promising treatment for stroke as well as an adjunctive agent to prevent its secondary complications.

Tang, Xian Nan; Cairns, Belinda; Kim, Jong Youl; Yenari, Midori A.

2013-01-01

78

Dopamine D1 receptor-mediated inhibition of NADPH oxidase activity in human kidney cells occurs via protein kinase A-protein kinase C cross-talk  

PubMed Central

Dopamine cellular signaling, via the D1 receptor (D1R), involves both protein kinase A (PKA) and protein kinase C (PKC), but the PKC isoform involved has not been determined. Therefore, we tested the hypothesis that the D1R-mediated inhibition of NADPH oxidase activity involves cross-talk between PKA and specific PKC isoform(s). In HEK-293 cells heterologously expressing human D1R (HEK-hD1), fenoldopam, a D1R agonist, and phorbol-12-myristate-13-acetate (PMA), a PKC activator, inhibited oxidase activity in a time- and concentration-dependent manner. The D1R-mediated inhibition of oxidase activity (68.1±3.6%) was attenuated by two different PKA inhibitors, H89 (10 µmol/L) (88±8.1%) and Rp-cAMP (10 µmol/L) (97.7±6.7%), and two different PKC inhibitors, bisindolylmaleimide I (1 µmol/L) (94±6%) and staurosporine (10 nmol/L) (93±8%), which by themselves, had no effect (n=4–8/group). The inhibitory effect of PMA (1 µmol/L) on oxidase activity (73±3.2%) was blocked by H89 (100±7.8%) (n=5–6/group). The PMA-mediated inhibition of NADPH oxidase activity was accompanied by an increase in PKC?S676, an effect that was also blocked by H89. Fenoldopam (1 µmol/L) also increased PKC?S676 in HEK-hD1 and human renal proximal tubule (RPT) cells. Knockdown of PKC? with siRNA in RPT cells prevented the inhibitory effect of fenoldopam on NADPH oxidase activity. Our studies demonstrate for the first time that cross-talk between PKA and PKC? plays an important role in the D1R-mediated negative regulation of NADPH oxidase activity in human kidney cells.

Yu, Peiying; Han, Weixing; Villar, Van Anthony M.; Li, Hewang; Arnaldo, Francis B.; Concepcion, Gisela P.; Felder, Robin A.; Quinn, Mark T.; Jose, Pedro A.

2011-01-01

79

D1Like Receptors Regulate NADPH Oxidase Activity and Subunit Expression in Lipid Raft Microdomains of Renal Proximal Tubule Cells  

Microsoft Academic Search

Abstract—NADPH oxidase (Nox)–dependent reactive oxygen,species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than

Hewang Li; Weixing Han; Anthony M. Villar; Lindsay B. Keever; Quansheng Lu; Ulrich Hopfer; Mark T. Quinn; Robin A. Felder; Pedro A. Jose; Peiying Yu

2010-01-01

80

Antimicrobial Actions of the NADPH Phagocyte Oxidase and Inducible Nitric Oxide Synthase in Experimental Salmonellosis. I. Effects on Microbial Killing by Activated Peritoneal Macrophages In Vitro  

Microsoft Academic Search

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL\\/6, congenic gp91 phox 2 \\/ 2 , iNOS 2 \\/ 2 , and doubly immunodeficient phox 2 \\/ 2 iNOS 2 \\/ 2 mice. The respiratory burst and

Andrés Vazquez-Torres; Jessica Jones-Carson; Pietro Mastroeni; Harry Ischiropoulos; Ferric C. Fang

81

Adverse cognitive effects of high-fat diet in a murine model of sleep apnea are mediated by NADPH oxidase activity.  

PubMed

Intermittent hypoxia (IH) during sleep, such as occurs in sleep apnea (SA), induces increased NADPH oxidase activation and deficits in hippocampal learning and memory. Similar to IH, high fat-refined carbohydrate diet (HFD), a frequent occurrence in patients with SA, can also induce similar oxidative stress and cognitive deficits under normoxic conditions, suggesting that excessive NADPH oxidase activity may underlie CNS dysfunction in both conditions. The effect of HFD and IH during the light period on two forms of spatial learning in the water maze as well as on markers of oxidative stress was assessed in male mice lacking NADPH oxidase activity (gp91phox?/Y) and wild-type littermates fed on HFD. On a standard place training task, gp91phox?/Y displayed normal learning, and was protected from the spatial learning deficits observed in wild-type littermates exposed to IH. Moreover, anxiety levels were increased in wild-type mice exposed to HFD and IH as compared to controls, while no changes emerged in gp91phox?/Y mice. Additionally, wild-type mice, but not gp91phox?/Y mice, had significantly elevated levels of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampal lysates following IH-HFD exposures. The cognitive deficits of obesity and westernized diets and those of sleep disorders that are characterized by IH during sleep are both mediated, at least in part, by excessive NADPH oxidase activity. PMID:23064009

Nair, D; Ramesh, V; Gozal, D

2012-10-11

82

Expression and activation of matrix metalloproteinase-9 and NADPH oxidase in tissues and plasma of experimental autoimmune encephalomyelitis in mice.  

PubMed

Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for multiple sclerosis (MS) that can be induced by immunization with myelin antigens such as myelin oligodendrocyte glycoprotein (MOG). The objective of this study was (i) to investigate how matrix metalloproteinase-9 (MMP-9) and NADPH oxidase enzymes are affected in the EAE mouse model and (ii) to know whether peripheral organs also express these enzymes in the EAE model. MOG(33-55) was administered subcutaneously on two sites over the back. Pertussis toxin was administered intraperitoneally immediately after MOG and again two days later. A significant difference was observed in body weights and clinical signs of EAE-induced mice. MMP-9 and NADPH oxidase enzymes were measured in central nervous system (CNS) tissues, peripheral tissues and plasma of EAE-induced mice. The primary findings include the distribution pattern of MMP-9 in CNS and peripheral tissues, and alterations in the enzymatic expression of MMP-9 and NADPH oxidase in the CNS tissues, spleen and plasma of EAE-induced mice. From these results, it can be considered that the spleen as well as the CNS can act as target organs in EAE disease, and plasma MMP-9 and NADPH oxidase may contribute to the pathogenesis of the disease. PMID:20810258

Kandagaddala, Lakshmi Devi; Kang, Min-Jung; Chung, Bong Chul; Patterson, Tucker A; Kwon, Oh-Seung

2010-08-31

83

A benzoxazine derivative induces vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 by elevating NADPH oxidase activity and reactive oxygen species levels.  

PubMed

Previously, we found that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO) promoted apoptosis of human umbilical vascular endothelial cells (HUVECs) deprived of growth factors. In this study, we aimed to investigate the effect of DBO and its mechanism of action on angiogenesis and apoptosis of HUVECs in the presence of fibroblast growth factor-2 (FGF-2), which promotes angiogenesis and inhibits apoptosis in vivo and in vitro. DBO significantly inhibited capillary-like tube formation by promoting apoptosis of HUVECs in the presence of FGF-2 in vitro. Furthermore, DBO elevated the levels of reactive oxygen species (ROS) and nitric oxide (NO) and increased the activity of NADPH oxidase and inducible nitric oxide synthase (iNOS) in promoting apoptosis under this condition. Moreover, when NADPH oxidase was inhibited by its specific inhibitor, dibenziodolium chloride (DPI), DBO could not elevate ROS and NO levels in HUVECs. The data suggest that DBO is a new modulator of apoptosis in vitro, and it might function by increasing the activity of NADPH oxidase and iNOS, subsequently elevating the levels of ROS and NO in HUVECs. The findings of this study provide a new small molecule for investigating the FGF-2/NADPH oxidase/iNOS signaling pathway in apoptosis. PMID:19539746

Zhao, Jing; He, Qiuxia; Cheng, Yizhe; Zhao, Baoxiang; Zhang, Yun; Zhang, Shangli; Miao, Junying

2009-06-17

84

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

85

The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities*  

PubMed Central

Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys59/Cys64), and a C-terminal cysteine/selenocysteine motif (Cys497/Sec498). TrxR has multiple roles in antioxidant defense. Opposing these functions, it may also assume a pro-oxidant role under some conditions. In the absence of its main electron-accepting substrates (e.g. thioredoxin), wild-type TrxR generates superoxide (O), which was here detected and quantified by ESR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The peroxidase activity of wild-type TrxR efficiently converted the O adduct (DEPMPO/HOO•) to the hydroxyl radical adduct (DEPMPO/HO•). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O. Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys59/Cys64 dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys59/Cys64 dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys497/Sec498 active site. Although the bulk of the DEPMPO/HO• signal generated by wild-type TrxR was due to its combined NADPH oxidase and Sec-dependent peroxidase activities, additional experiments showed that some free HO• could be generated by the enzyme in an H2O2-dependent and Sec-independent manner. The direct NADPH oxidase and peroxidase activities of TrxR characterized here give insights into the full catalytic potential of this enzyme and may have biological consequences beyond those solely related to its reduction of thioredoxin.

Cheng, Qing; Antholine, William E.; Myers, Judith M.; Kalyanaraman, Balaraman; Arner, Elias S. J.; Myers, Charles R.

2010-01-01

86

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.

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

2012-01-01

87

NADPH oxidase as a therapeutic target in Alzheimer's disease  

PubMed Central

At present, available treatments for Alzheimer's disease (AD) are largely unable to halt disease progression. Microglia, the resident macrophages in the brain, are strongly implicated in the pathology and progressively degenerative nature of AD. Specifically, microglia are activated in response to both ? amyloid (A?) and neuronal damage, and can become a chronic source of neurotoxic cytokines and reactive oxygen species (ROS). NADPH oxidase is a multi-subunit enzyme complex responsible for the production of both extracellular and intracellular ROS by microglia. Importantly, NADPH oxidase expression is upregulated in AD and is an essential component of microglia-mediated A? neurotoxicity. Activation of microglial NADPH oxidase causes neurotoxicity through two mechanisms: 1) extracellular ROS produced by microglia are directly toxic to neurons; 2) intracellular ROS function as a signaling mechanism in microglia to amplify the production of several pro-inflammatory and neurotoxic cytokines (for example, tumor necrosis factor-?, prostaglandin E2, and interleukin-1?). The following review describes how targeting NADPH oxidase can reduce a broad spectrum of toxic factors (for example, cytokines, ROS, and reactive nitrogen species) to result in inhibition of neuronal damage from two triggers of deleterious microglial activation (A? and neuron damage), offering hope in halting the progression of AD.

Block, Michelle L

2008-01-01

88

Novel isoforms of NADPH oxidase in vascular physiology and pathophysiology.  

PubMed

1. Vascular cells have evolved to use reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, as signalling molecules. Under physiological conditions, ROS are important regulators of cell cycle, protein kinase activity and gene expression. However, in vascular disease states, such as hypertension and hypercholesterolaemia, excessive production of ROS may overwhelm the anti-oxidant defence mechanisms of cells, resulting in 'oxidative stress', damage to the artery wall and, ultimately, development of atherosclerotic plaques. 2. The primary source of ROS in the vasculature is NADPH oxidase. There appear to be at least three isoforms of NADPH oxidase expressed in the vascular wall, each differing with respect to the flavin-containing catalytic subunit it uses to transfer electrons from NADPH to molecular oxygen. Thus, although endothelial cells and adventitial fibroblasts express a gp91phox-containing NADPH oxidase similar to that originally identified in phagocytes, vascular smooth muscle cells may rely on novel homologues of gp91phox, namely Nox1 and Nox4, to produce superoxide. 3. Controversy remains over which isoform(s) of NADPH oxidase is responsible for the oxidative stress associated with vascular diseases. We and others have shown that although gp91phox mRNA expression is upregulated during atherogenesis in human and animal models, expression of the Nox4 subunit remains unchanged. Nox1 expression is also likely to be increased in diseased arteries; however, its relative level of expression, at least at the mRNA level, appears to be markedly lower than that of the other gp91phox homologues, even after upregulation. 4. Whether these findings suggest that a gp91phox-containing NADPH oxidase is more important than either Nox4 or Nox1 in vascular disease awaits studies examining relative protein expression and enzyme kinetics of each subunit, as well as the effects of targeted gene deletion of each of these gp91phox homologues on atherogenesis. PMID:14678249

Bengtsson, Sara H M; Gulluyan, Lerna M; Dusting, Gregory J; Drummond, Grant R

2003-11-01

89

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.

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

2011-01-01

90

Phospholipase D?1 and Phosphatidic Acid Regulate NADPH Oxidase Activity and Production of Reactive Oxygen Species in ABA-Mediated Stomatal Closure in Arabidopsis[C][W][OA  

PubMed Central

We determined the role of Phospholipase D?1 (PLD?1) and its lipid product phosphatidic acid (PA) in abscisic acid (ABA)-induced production of reactive oxygen species (ROS) in Arabidopsis thaliana guard cells. The pld?1 mutant failed to produce ROS in guard cells in response to ABA. ABA stimulated NADPH oxidase activity in wild-type guard cells but not in pld?1 cells, whereas PA stimulated NADPH oxidase activity in both genotypes. PA bound to recombinant Arabidopsis NADPH oxidase RbohD (respiratory burst oxidase homolog D) and RbohF. The PA binding motifs were identified, and mutation of the Arg residues 149, 150, 156, and 157 in RbohD resulted in the loss of PA binding and the loss of PA activation of RbohD. The rbohD mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure. Furthermore, ABA-induced production of nitric oxide (NO) was impaired in pld?1 guard cells. Disruption of PA binding to ABI1 protein phosphatase 2C did not affect ABA-induced production of ROS or NO, but the PA–ABI1 interaction was required for stomatal closure induced by ABA, H2O2, or NO. Thus, PA is as a central lipid signaling molecule that links different components in the ABA signaling network in guard cells.

Zhang, Yanyan; Zhu, Huiying; Zhang, Qun; Li, Maoyin; Yan, Min; Wang, Rong; Wang, Liling; Welti, Ruth; Zhang, Wenhua; Wang, Xuemin

2009-01-01

91

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

92

TNF-alpha dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation.  

PubMed

Expression of TNF-alpha, a pleiotropic cytokine, is elevated during stroke and cerebral ischemia. TNF-alpha regulates arterial diameter, although mechanisms mediating this effect are unclear. In the present study, we tested the hypothesis that TNF-alpha regulates the diameter of resistance-sized ( approximately 150-microm diameter) cerebral arteries by modulating local and global intracellular Ca(2+) signals in smooth muscle cells. Laser-scanning confocal imaging revealed that TNF-alpha increased Ca(2+) spark and Ca(2+) wave frequency but reduced global intracellular Ca(2+) concentration ([Ca(2+)](i)) in smooth muscle cells of intact arteries. TNF-alpha elevated reactive oxygen species (ROS) in smooth muscle cells of intact arteries, and this increase was prevented by apocynin or diphenyleneiodonium (DPI), both of which are NAD(P)H oxidase blockers, but was unaffected by inhibitors of other ROS-generating enzymes. In voltage-clamped (-40 mV) cells, TNF-alpha increased the frequency and amplitude of Ca(2+) spark-induced, large-conductance, Ca(2+)-activated K(+) (K(Ca)) channel transients approximately 1.7- and approximately 1.4-fold, respectively. TNF-alpha-induced transient K(Ca) current activation was reversed by apocynin or by Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a membrane-permeant antioxidant, and was prevented by intracellular dialysis of catalase. TNF-alpha induced reversible and similar amplitude dilations in either endothelium-intact or endothelium-denuded pressurized (60 mmHg) cerebral arteries. MnTMPyP, thapsigargin, a sarcoplasmic reticulum Ca(2+)-ATPase blocker that inhibits Ca(2+) sparks, and iberiotoxin, a K(Ca) channel blocker, reduced TNF-alpha-induced vasodilations to between 15 and 33% of control. In summary, our data indicate that TNF-alpha activates NAD(P)H oxidase, resulting in an increase in intracellular H(2)O(2) that stimulates Ca(2+) sparks and transient K(Ca) currents, leading to a reduction in global [Ca(2+)](i), and vasodilation. PMID:16267103

Cheranov, Sergey Y; Jaggar, Jonathan H

2005-11-02

93

Modulation of cellular redox status by thiamine-activated NADPH oxidase confers Arabidopsis resistance to Sclerotinia sclerotiorum.  

PubMed

Sclerotinia sclerotiorum can initially suppress host oxidative burst to aid infection establishment, but later promotes reactive oxygen species (ROS) generation as proliferation advances. Here, it was shown that the cellular redox status can be modulated by thiamine to protect Arabidopsis thaliana against Sclerotinia at the early stages of infection. The initial inhibition of host ROS generation by Sclerotinia-secreted oxalate could effectively be alleviated by thiamine. Thiamine pre-treatment and subsequent wild-type Sclerotinia invasion induced an increase of ascorbate peroxidase activity concomitant with decreased ascorbate/dehydroascorbate ratios, which led to the cellular transition towards oxidative status in infected tissues. Particularly, it was observed that wild-type Sclerotinia, but not oxalate-deficient A2 mutant, could suppress the activity of NADPH oxidase (NOX), which might be an important mechanism underlying the early inhibition of ROS burst. Nevertheless, thiamine pre-treatment followed by wild-type Sclerotinia infection promoted NOX-derived ROS accumulation. Further studies showed that cytosolic Ca(2+) and staurosporine-sensitive protein kinase(s) participated in thiamine-induced activation of NOX. Moreover, thiamine-induced tissue defence responses including callose/lignin deposition and stomatal closure were closely correlated with NOX-derived ROS generation. Additionally, studies with Brassica species indicated that the regulation of thiamine is largely conserved upon Sclerotinia infection. Collectively, it was concluded that thiamine reverses the initial reducing status through activating NOX-dependent ROS signalling to perturb the disease progress of Sclerotinia. PMID:23814275

Zhou, Jun; Sun, Aizhen; Xing, Da

2013-06-27

94

[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

95

Renalase Is an ?-NAD(P)H Oxidase/Anomerase.  

PubMed

Renalase is a protein hormone secreted into the blood by the kidney that is reported to lower blood pressure and slow heart rate. Since its discovery in 2005, renalase has been the subject of conjecture pertaining to its catalytic function. While it has been widely reported that renalase is the third monoamine oxidase (monoamine oxidase C) that oxidizes circulating catecholamines such as epinephrine, there has been no convincing demonstration of this catalysis in vitro. Renalase is a flavoprotein whose structural topology is similar to known oxidases, lysine demethylases, and monooxygenases, but its active site bears no resemblance to that of any known flavoprotein. We have identified the catalytic activity of renalase as an ?-NAD(P)H oxidase/anomerase, whereby low equilibrium concentrations of the ?-anomer of NADPH and NADH initiate rapid reduction of the renalase flavin cofactor. The reduced cofactor then reacts with dioxygen to form hydrogen peroxide and releases nicotinamide dinucleotide product in the ?-form. These processes yield an apparent turnover number (0.5 s(-1) in atmospheric dioxygen) that is at least 2 orders of magnitude more rapid than any reported activity with catechol neurotransmitters. This highly novel activity is the first demonstration of a role for naturally occurring ?-NAD(P)H anomers in mammalian physiology and the first report of a flavoprotein catalyzing an epimerization reaction. PMID:23964689

Beaupre, Brett A; Carmichael, Brenton R; Hoag, Matthew R; Shah, Dhara D; Moran, Graham R

2013-09-05

96

Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Absolute lipid dependence of the solubilized enzyme.  

PubMed

The superoxide (O2-)-forming NADPH oxidase of resting macrophages can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). O2- production requires the cooperation of membrane-associated and cytosolic components. The membrane component can be solubilized by octyl glucoside yielding a highly active oxidase preparation. High performance gel filtration of the solubilized oxidase on Superose 12 in the presence of 40 mM octyl glucoside leads to the total loss of enzymatic activity. This can be restored in previously inactive eluate fractions by "reconstitution" with N-ethylmaleimide or heat (60 degrees C)-inactivated total solubilized membrane. Oxidase activity, that becomes evident upon reconstitution, is eluted from Superose 12 with molecules in the Mr range of 300,000-71,000. The material with reconstitutive capacity is completely dissociated from the oxidase, eluting with molecules in the Mr range of 71,000-11,000. The Superose 12 elution profile of the material responsible for reconstitution coincides with that of membrane-derived phospholipid. Also, the reconstitutive capacity of total solubilized membrane and that of the Mr 71,000-11,000 region of the Superose eluate are recovered in a chloroform extract prepared by the method of Bligh and Dyer. It is concluded that loss of oxidase activity by gel filtration at a high octyl glucoside concentration is the consequence of delipidation. NADPH oxidase activity, revealed by reconstitution of Superose 12 fractions with exogenous phospholipid, correlates closely with the elution profile of cytochrome b559. Reconstitution of activity of delipidated oxidase can also be achieved with natural non-macrophage phospholipids and with synthetic phospholipids. Reconstitution of NADPH oxidase activity by lipids is governed by the following rules: (a) phospholipids are effective; lysophospholipids and neutral lipids are not; (b) phospholipids with polar heads represented by choline, ethanolamine, and serine, as well as cardiolipin, are effective; phosphatidylinositol is much less active; (c) phospholipids with unsaturated fatty acid residues are capable of reconstitution while saturated acyl residues do not confer activity; this specificity appears not to be related to the transition temperature of the phospholipids. PMID:2542302

Shpungin, S; Dotan, I; Abo, A; Pick, E

1989-06-01

97

The role of NADPH oxidase in carotid body arterial chemoreceptors  

PubMed Central

O2-sensing in the carotid body occurs in neuroectoderm-derived type I glomus cells where hypoxia elicits a complex chemotransduction cascade involving membrane depolarization, Ca2+ entry and the release of excitatory neurotransmitters. Efforts to understand the exquisite O2-sensitivity of these cells currently focus on the coupling between local PO2 and the open-closed state of K+-channels. Amongst multiple competing hypotheses is the notion that K+-channel activity is mediated by a phagocytic-like multisubunit enzyme, NADPH oxidase, which produces reactive oxygen species (ROS) in proportion to the prevailing PO2. In O2-sensitive cells of lung neuroepithelial bodies (NEB), multiple studies confirm that ROS levels decrease in hypoxia, and that EM and K+-channel activity are indeed controlled by ROS produced by NADPH oxidase. However, recent studies in our laboratories suggest that ROS generated by a non-phagocyte isoform of the oxidase are important contributors to chemotransduction, but that their role in type I cells differs fundamentally from the mechanism utilized by NEB chemoreceptors. Data indicate that in response to hypoxia, NADPH oxidase activity is increased in type I cells, and further, that increased ROS levels generated in response to low-O2 facilitate cell repolarization via specific subsets of K+-channels.

Dinger, B.; He, L.; Chen, J.; Liu, X.; Gonzalez, C.; Obeso, A.; Sanders, K.; Hoidal, J.; Stensaas, L.; Fidone, S.

2007-01-01

98

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

99

NADPH oxidase activity controls phagosomal proteolysis in macrophages through modulation of the lumenal redox environment of phagosomes  

PubMed Central

The phagosomal lumen in macrophages is the site of numerous interacting chemistries that mediate microbial killing, macromolecular degradation, and antigen processing. Using a non-hypothesis-based screen to explore the interconnectivity of phagosomal functions, we found that NADPH oxidase (NOX2) negatively regulates levels of proteolysis within the maturing phagosome of macrophages. Unlike the NOX2 mechanism of proteolytic control reported in dendritic cells, this phenomenon in macrophages is independent of changes to lumenal pH and is also independent of hydrolase delivery to the phagosome. We found that NOX2 mediates the inhibition of phagosomal proteolysis in macrophages through reversible oxidative inactivation of local cysteine cathepsins. We also show that NOX2 activity significantly compromises the phagosome's ability to reduce disulfides. These findings indicate that NOX2 oxidatively inactivates cysteine cathepsins through sustained ablation of the reductive capacity of the phagosomal lumen. This constitutes a unique mechanism of spatiotemporal control of phagosomal chemistries through the modulation of the local redox environment. In addition, this work further implicates the microbicidal effector NOX2 as a global modulator of phagosomal physiologies, particularly of those pertinent to antigen processing.

Rybicka, Joanna M.; Balce, Dale R.; Khan, Morgan F.; Krohn, Regina M.; Yates, Robin M.

2010-01-01

100

Involvement of NADPH oxidase in age-associated cardiac remodeling  

Microsoft Academic Search

Increased activation of the renin–angiotensin–aldosterone system (RAAS) and an increase in oxidative stress are both implicated in age-related cardiac remodeling but their precise interrelationship and linkage to underlying molecular and cellular abnormalities remain to be defined. Recent studies indicate that NADPH oxidases are major sources of oxidative stress and are activated by the RAAS. This study investigated the relationship between

Mingyi Wang; Jing Zhang; Simon J. Walker; Rafal Dworakowski; Edward G. Lakatta; Ajay M. Shah

2010-01-01

101

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

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 Ca(2+) concentration ([Ca(2+)](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 p47(phox) and p67(phox) to the cell membrane and was necessary for ROS generation by Khz. Khz triggered a rapid and sustained increase in [Ca(2+)](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 p47(phox) and p67(phox) 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 [Ca(2+)](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

2012-10-08

102

Adenosine A1 receptor activation is arrhythmogenic in the developing heart through NADPH oxidase/ERK- and PLC/PKC-dependent mechanisms.  

PubMed

Whether adenosine, a crucial regulator of the developing cardiovascular system, can provoke arrhythmias in the embryonic/fetal heart remains controversial. Here, we aimed to establish a mechanistic basis of how an adenosinergic stimulation alters function of the developing heart. Spontaneously beating hearts or dissected atria and ventricle obtained from 4-day-old chick embryos were exposed to adenosine or specific agonists of the receptors A(1)AR (CCPA), A(2A)AR (CGS-21680) and A(3)AR (IB-MECA). Expression of the receptors was determined by quantitative PCR. The functional consequences of blockade of NADPH oxidase, extracellular signal-regulated kinase (ERK), phospholipase C (PLC), protein kinase C (PKC) and L-type calcium channel (LCC) in combination with adenosine or CCPA, were investigated in vitro by electrocardiography. Furthermore, the time-course of ERK phosphorylation was determined by western blotting. Expression of A(1)AR, A(2A)AR and A(2B)AR was higher in atria than in ventricle while A(3)AR was equally expressed. Adenosine (100?M) triggered transient atrial ectopy and second degree atrio-ventricular blocks (AVB) whereas CCPA induced mainly Mobitz type I AVB. Atrial rhythm and atrio-ventricular propagation fully recovered after 60min. These arrhythmias were prevented by the specific A(1)AR antagonist DPCPX. Adenosine and CCPA transiently increased ERK phosphorylation and induced arrhythmias in isolated atria but not in ventricle. By contrast, A(2A)AR and A(3)AR agonists had no effect. Interestingly, the proarrhythmic effect of A(1)AR stimulation was markedly reduced by inhibition of NADPH oxidase, ERK, PLC, PKC or LCC. Moreover, NADPH oxidase inhibition or antioxidant MPG prevented both A(1)AR-mediated arrhythmias and ERK phosphorylation. These results suggest that pacemaking and conduction disturbances are induced via A(1)AR through concomitant stimulation of NADPH oxidase and PLC, followed by downstream activation of ERK and PKC with LCC as possible target. PMID:21907719

Robin, Elodie; Sabourin, Jessica; Benoit, Rachel; Pedretti, Sarah; Raddatz, Eric

2011-08-31

103

NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-?-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells.  

PubMed

TNF-? plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-? in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-?-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-? induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-?-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-l-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-?B (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47(phox), p42, p38, JNK1, p65, or PYK2. Moreover, TNF-? markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-?-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-?B (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-?-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-?-stimulated MAPKs and NF-?B activation. Thus, in H9c2 cells, we are the first to show that TNF-?-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-?B cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-?-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-? on H9c2 cells may provide potential therapeutic targets of chronic heart failure. PMID:23774252

Yang, Chuen-Mao; Lee, I-Ta; Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der

2013-06-14

104

Role of NADPH oxidase in host defense against aspergillosis.  

PubMed

NADPH oxidase plays a critical role in antimicrobial host defense, as evident in chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase characterized by severe bacterial and fungal diseases. Invasive aspergillosis and other moulds are the major cause of mortality in CGD. We also learn from CGD patients that NADPH oxidase plays an important role in regulating inflammation; CGD patients are prone to developing inflammatory diseases such as inflammatory bowel disease, obstructive granulomata of the genitourinary tract, and hypersensitivity pneumonitis. Indeed, the NADPH oxidase plays an essential role in calibrating innate and T-cell responses to control the growth of inhaled fungi while protecting against excessive and injurious inflammation. Knowledge gained on the mechanisms by which NADPH oxidase kills fungi and regulates inflammation may lead to new therapeutics for CGD and will have broad relevance to understanding host-pathogen interactions between mammals and ubiquitous moulds to which we are continually exposed. PMID:20560866

Grimm, Melissa J; Vethanayagam, R Robert; Almyroudis, Nikolaos G; Lewandowski, David; Rall, Nicole; Blackwell, Timothy S; Segal, Brahm H

2010-06-21

105

Monocyte- and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice.  

PubMed

Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate ?-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation. PMID:23509361

Grimm, Melissa J; Vethanayagam, R Robert; Almyroudis, Nikolaos G; Dennis, Carly G; Khan, A Nazmul H; D'Auria, Anthony C; Singel, Kelly L; Davidson, Bruce A; Knight, Paul R; Blackwell, Timothy S; Hohl, Tobias M; Mansour, Michael K; Vyas, Jatin M; Röhm, Marc; Urban, Constantin F; Kelkka, Tiina; Holmdahl, Rikard; Segal, Brahm H

2013-03-15

106

The membrane-associated component of the amphiphile-activated, cytosol-dependent superoxide-forming NADPH oxidase of macrophages is identical to cytochrome b559.  

PubMed

The superoxide (O2-) forming NADPH oxidase complex of resting phagocytes can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). For O2- production to occur, the participation of both membrane-associated and cytosol-derived components is required. The purpose of this investigation was to isolate and characterize the membrane component of NADPH oxidase. For this purpose, guinea pig macrophage membranes were extracted with 1 M NaCl, solubilized by 40 mM octyl glucoside, and subjected to a purification sequence consisting of absorption with DEAE-Sepharose, affinity chromatography on heparin-agarose, and chromatography on hydroxylapatite. At each purification step, fractions were assayed for their ability to support SDS-elicited, cytosol-dependent O2- production, following incorporation in liposomes of phosphatidylcholine. We found that membrane oxidase activity copurified strictly with cytochrome b559. Peak hydroxylapatite fractions exhibited specific O2(-)-forming activity in the range of 81-115 mumol of O2-/mg protein/min and a specific cytochrome b559 content of 7-14 nmol of cytochrome b559/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the peak oxidase activity fractions, derived by hydroxylapatite chromatography, revealed essentially two bands that were identified as the beta (54-60 kDa) and alpha (21/22 kDa) subunits of guinea pig cytochrome b559. The relation of the two polypeptides to cytochrome b559 was established by correlation with a spectral signal characteristic of cytochrome b559, immunoblotting with antibodies against defined human cytochrome b559 beta and alpha chain peptides, cross-linking studies, and deglycosylation experiments. Hydroxylapatite-purified membrane oxidase preparations did not contain FAD and were free of cytochrome c reductase activity. Purified membrane oxidase preparations were also capable of cooperating with purified cytosolic components in SDS-elicited cell-free O2- production. We conclude that the membrane-associated component of the O2- generating NADPH oxidase is identical to cytochrome b559. PMID:1847135

Knoller, S; Shpungin, S; Pick, E

1991-02-15

107

NADPH Oxidase Priming and p47phox Phosphorylation in Neutrophils from Synovial Fluid of Patients with Rheumatoid Arthritis and Spondylarthropathy  

Microsoft Academic Search

Superoxide anion (O2°-)production by neutrophil NADPH oxidase participates in arthritic joint lesion formation. Proinflammatory cytokines such as tumor necrosis factor a(TNFa), interleukin 8 (IL-8) and granulocyte\\/macrophage-colony stimulating factor (GM-CSF) have a priming effect on neutrophil NADPH oxidase activity. NADPH oxidase activation is dependent on phosphorylation of p47phox, a cytosolic component of the enzyme. We studied O2°-production and p47phox phosphorylation in

JamelEl Benna; Gilles Hayem; PhamMy-Chan Dang; Michčle Fay; Sylvie Chollet-Martin; Carole Elbim; Olivier Meyer; Marie-Anne Gougerot-Pocidalo

2002-01-01

108

Synergistic Activation of Glucose-6-Phosphate Dehydrogenase and NAD(P)H oxidase by Src kinase Elevates Superoxide in Type 2 Diabetic, Zucker fa/fa, Rat Liver  

PubMed Central

Glucose metabolism through glycolysis and hexosamine pathway has been shown to be altered in type 2 diabetes. However, its fate through the pentose phosphate pathway (PPP) is currently unclear. In this study, we determined whether the activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the PPP, is modulated in the liver of Zucker obese fa/fa rats (9–11 weeks old). We found that G6PD expression and activity, NADPH levels and 6-phosphogluconate generation was significantly increased in liver of fa/fa rats. Inhibition of PI3 kinase and Src kinases decreased (P<0.05) G6PD activity in the fa/fa but not in the lean rat liver, suggesting that G6PD activity is regulated by PI3/Src kinase signaling pathways. G6PD-derived NADPH increased (P<0.05) superoxide anion levels by 70–90% in the fa/fa vs the lean rat liver, which was inhibited by NADPH oxidase inhibitor, gp91ds-tat (50 ?M), and G6PD inhibitors, 6-aminonicotinamide (1 mM) and dihydroepiandrosterone (100 ?M); therefore, indicating that elevated G6PD activity may be responsible for mediating superoxide generation. Interestingly, we also found a positive correlation between liver hypertrophy/increased G6PD activity (r2=0.77; P=0.0009) and liver hypertrophy/superoxide production (r2=0.51; P=0.0091) in fa/fa rats. Increased G6PD and NADPH oxidase expression and activity, in young hyper-glycemic and -insulinemic rats prior to the development of diabetes, appear to be contributing factors for the induction of oxidative stress. Since inhibition of G6PD activity decreases oxidative stress, we conclude that it behaves as a pro-oxidant in the fa/fa rat liver, in type 2 diabetes.

Gupte, Rakhee S.; Floyd, Beverly C.; Kozicky, Mark; George, Shimran; Ungvari, Zoltan I.; Neito, Vanessa; Wolin, Michael S.; Gupte, Sachin A

2009-01-01

109

Antimicrobial Actions of the Nadph Phagocyte Oxidase and Inducible Nitric Oxide Synthase in Experimental Salmonellosis. I. Effects on Microbial Killing by Activated Peritoneal Macrophages in Vitro  

PubMed Central

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox?/?, iNOS?/?, and doubly immunodeficient phox?/?iNOS?/? mice. The respiratory burst and NO radical (NO·) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase–dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O2·?), hydrogen peroxide (H2O2), and peroxynitrite (ONOO?) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S. typhimurium. Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon ? appears to augment antibacterial activity predominantly by enhancing NO· production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

Vazquez-Torres, Andres; Jones-Carson, Jessica; Mastroeni, Pietro; Ischiropoulos, Harry; Fang, Ferric C.

2000-01-01

110

Regulation of NAD(P)H Oxidase Growth and Transcription in Melanoma Cells.  

National Technical Information Service (NTIS)

Malignant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-kB (NF-kB). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constit...

T. P. Kennedy

2002-01-01

111

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.

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

1996-01-01

112

Reactive oxygen species and NADPH oxidase 4 induced by transforming growth factor ?1 are the therapeutic targets of polyenylphosphatidylcholine in the suppression of human hepatic stellate cell activation  

Microsoft Academic Search

Objective and design  To clarify the molecular mechanism of polyenylphosphatidylcholine (PPC), we examined the involvement of reactive oxygen species\\u000a (ROS) and NADPH oxidase 4 (Nox4) in human hepatic stellate cells (HSCs).\\u000a \\u000a \\u000a \\u000a \\u000a Material  Using human LX-2 HSC cells, we examined the effects of PPC on expression of ?-smooth muscle actin (?-SMA) and collagen 1,\\u000a generation of ROS, Nox4 expression, p38 activation and cell

Remina Ikeda; Kyoko Ishii; Yoshiko Hoshikawa; Junya Azumi; Yuta Arakaki; Toshihiro Yasui; Shizuka Matsuura; Yoshiaki Matsumi; Yohei Kono; Yusuke Mizuta; Akihiro Kurimasa; Ichiro Hisatome; Scott L. Friedman; Hironaka Kawasaki; Goshi Shiota

2011-01-01

113

APE1/Ref-1 promotes the effect of angiotensin II on Ca2+ -activated K+ channel in human endothelial cells via suppression of NADPH oxidase.  

PubMed

The effects of angiotensin II (Ang II) on whole-cell large conductance Ca(2+)-activated K(+) (BK(Ca)) currents was investigated in control and Apurinic/apyrimidinic endonuclease1/redox factor 1 (APE1/Ref-1)-overexpressing human umbilical vein endothelial cells (HUVECs). Ang II blocked the BK(Ca) current in a dose-dependent fashion, and this inhibition was greater in APE1/Ref-1-overexpressing HUVECs than in control HUVECs (half-inhibition values of 102.81+/-9.54 nM and 11.34+/-0.39 nM in control and APE1/Ref-1-overexpressing HUVECs, respectively). Pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) or knock down of NADPH oxidase (p22 phox) using siRNA increased the inhibitory effect of Ang II on the BK(Ca) currents, similar to the effect of APE1/Ref-1 overexpression. In addition, application of Ang II increased the superoxide and hydrogen peroxide levels in the control HUVECs but not in APE1/Ref-1-overexpressing HUVECs. Furthermore, direct application of hydrogen peroxide increased BK(Ca) channel activity. Finally, the inhibitory effect of Ang II on the BK(Ca) current was blocked by an antagonist of the Ang II type 1 (AT(1)) receptor in both control and APE1/Ref-1-overexpressing HUVECs. From these results, we conclude that the inhibitory effect of Ang II on BK(Ca) channel function is NADPH oxidase-dependent and may be promoted by APE1/Ref-1. PMID:18958420

Park, Won Sun; Ko, Eun A; Jung, In Duk; Son, Youn Kyoung; Kim, Hyoung Kyu; Kim, Nari; Park, So Youn; Hong, Ki Whan; Park, Yeong-Min; Choi, Tae-Hoon; Han, Jin

2008-10-29

114

The NADPH oxidase of professional phagocytes—prototype of the NOX electron transport chain systems  

Microsoft Academic Search

The NADPH oxidase is an electron transport chain in “professional” phagocytic cells that transfers electrons from NADPH in the cytoplasm, across the wall of the phagocytic vacuole, to form superoxide. The electron transporting flavocytochrome b is activated by the integrated function of four cytoplasmic proteins. The antimicrobial function of this system involves pumping K+ into the vacuole through BKCa channels,

Andrew R Cross; Anthony W Segal

2004-01-01

115

NAD(P)H Oxidase Role in Cardiovascular Biology and Disease  

Microsoft Academic Search

Reactive oxygen species have emerged as important molecules in cardiovascular function. Recent work has shown that NAD(P)H oxidases are major sources of superoxide in vascular cells and myocytes. The biochemical characterization, activation paradigms, structure, and function of this enzyme are now partly understood. Vascular NAD(P)H oxidases share some, but not all, characteristics of the neutrophil enzyme. In response to growth

Kathy K. Griendling; Dan Sorescu; Masuko Ushio-Fukai; David G. Harrison

116

Dietary sesamin suppresses aortic NADPH oxidase in DOCA salt hypertensive rats.  

PubMed

1. Dietary sesamin, a sesame lignan, is known to suppress the development of experimental hypertension in rats partly through its inhibitory effect on vascular O(2)(-) production. Therefore, in the present study, we examined whether sesamin feeding had any effect on vascular NADPH oxidase using aortas from deoxycorticosterone acetate (DOCA) salt hypertensive rats. 2. After a 5 week feeding and treatment period, aortic O(2)(-) production and NADPH oxidase activity were measured using the lucigenin assay. Reverse transcription-polymerase chain reaction was performed to analyse aortic expression of NADPH oxidase subunit (p22phox, gp91phox, Nox1 and Nox4) mRNA. 3. Sesamin feeding markedly suppressed DOCA salt-induced hypertension and significantly decreased aortic O(2)(-) production. DOCA salt treatment increased NADPH oxidase activity and elevated aortic mRNA expression of p22phox, gp91phox, Nox1 and Nox4. Sesamin feeding abolished the increase in NADPH oxidase activity and, furthermore, significantly suppressed increases in p22phox, gp91phox and Nox1 mRNA expression. 4. In conclusion, dietary sesamin prevented DOCA salt-induced increases in NADPH oxidase activity and subunit mRNA expression. These effects seem to be involved in the anti-oxidant and antihypertensive effects of sesamin. PMID:17941888

Nakano, Daisuke; Kurumazuka, Daisuke; Nagai, Yukiko; Nishiyama, Akira; Kiso, Yoshinobu; Matsumura, Yasuo

2007-10-17

117

Lipid rafts keep NADPH oxidase in the inactive state in human renal proximal tubule cells.  

PubMed

Recent studies have indicated the importance of cholesterol-rich membrane lipid rafts (LRs) in oxidative stress-induced signal transduction. Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases, the major sources of reactive oxygen species, are implicated in cardiovascular diseases, including hypertension. We tested the hypothesis that NADPH oxidase subunits and activity are regulated by LRs in human renal proximal tubule cells. We report that a high proportion of p22(phox) and the small GTPase Rac1 are expressed in LRs in human renal proximal tubule cells. The D(1)-like receptor agonist, fenoldopam (1 micromol/L per 20 minutes) dispersed Nox subunits within LRs and non-LRs and decreased oxidase activity (30.7+/-3.3%). In contrast, cholesterol depletion (2% methyl-beta-cyclodextrin [beta CD]) translocated NADPH oxidase subunits out of LRs and increased oxidase activity (154.0+/-10.5% versus control, 103.1+/-3.4%), which was reversed by cholesterol repletion (118.9+/-9.9%). Moreover, NADPH oxidase activation by beta CD (145.5+/-9.0%; control: 98.6+/-1.6%) was also abrogated by the NADPH oxidase inhibitors apocynin (100.4+/-3.2%) and diphenylene iodonium (9.5+/-3.3%). Furthermore, beta CD-induced reactive oxygen species production was reversed by knocking down either Nox2 (81.0+/-5.1% versus beta CD: 162.0+/-2.0%) or Nox4 (108.0+/-10.8% versus beta CD: 152.0+/-9.8%). We have demonstrated for the first time that disruption of LRs results in NADPH oxidase activation that is abolished by antioxidants and silencing of Nox2 or Nox4. Therefore, in human renal proximal tubule cells, LRs maintain NADPH oxidase in an inactive state. PMID:18195159

Han, Weixing; Li, Hewang; Villar, Van Anthony M; Pascua, Annabelle M; Dajani, Mustafa I; Wang, Xiaoyang; Natarajan, Aruna; Quinn, Mark T; Felder, Robin A; Jose, Pedro A; Yu, Peiying

2008-01-14

118

Induction of heme oxygenase-1 in vivo suppresses NADPH oxidase derived oxidative stress.  

PubMed

Our previous studies suggest that heme oxygenase (HO)-1 induction and/or subsequent bilirubin generation in endothelial cells may suppress superoxide generation of from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. In this study, we examined the consequence of HO-1 induction in vivo on NADPH oxidase activity. Three doses of hemin (25 mg x kg(-1), IP, every 48 hours), with or without cotreatment with the HO inhibitor tin protoporphyrin-IX (15 mg x kg(-1), IP), were given to apolipoprotein E-deficient mice, which display vascular oxidative stress. Hemin treatment increased HO-1 expression and activity in aorta (undetectable at baseline) and kidney (by 3-fold) and significantly reduced both NADPH oxidase activity (by approximately 25% to 50%) and superoxide generation in situ. The increase in HO-1 activity and inhibition of NADPH oxidase activity by hemin were reversed by tin protoporphyrin-IX and were not associated with changes in Nox2 or Nox4 protein levels. Hemin also reduced plasma F(2)-isoprostane levels by 23%. The inhibition of NADPH oxidase activity by hemin in the aorta was mimicked by bilirubin in vitro (0.01 to 1 micromol/L). Bilirubin also concentration-dependently reduced NADPH oxidase-dependent superoxide production stimulated by angiotensin II in rat vascular smooth muscle cells and by phorbol 12-myristate 13-acetate in human neutrophil-like HL-60 cells. HO-1 overexpression by plasmid-mediated gene transfer in rat vascular smooth muscle cells decreased NADPH-stimulated superoxide production. Thus, systemic expression of HO-1 suppresses NADPH oxidase activity by mechanisms at least partly mediated by the bile pigment bilirubin, thereby reducing oxidative stress. PMID:17679649

Datla, Srinivasa R; Dusting, Gregory J; Mori, Trevor A; Taylor, Caroline J; Croft, Kevin D; Jiang, Fan

2007-08-06

119

Blockade of TGF-? 1 signalling inhibits cardiac NADPH oxidase overactivity in hypertensive rats.  

PubMed

NADPH oxidases constitute a major source of superoxide anion (·O(2)(-)) in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-? 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-? 1 receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR) to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-? 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-? 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-? 1. PMID:22701756

Miguel-Carrasco, José Luis; Baltanás, Ana; Cebrián, Carolina; Moreno, María U; López, Begońa; Hermida, Nerea; González, Arantxa; Dotor, Javier; Borrás-Cuesta, Francisco; Díez, Javier; Fortuńo, Ana; Zalba, Guillermo

2012-06-03

120

Blockade of TGF-? 1 Signalling Inhibits Cardiac NADPH Oxidase Overactivity in Hypertensive Rats  

PubMed Central

NADPH oxidases constitute a major source of superoxide anion (·O2??) in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-? 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-? 1 receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR) to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-? 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-? 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-? 1.

Miguel-Carrasco, Jose Luis; Baltanas, Ana; Cebrian, Carolina; Moreno, Maria U.; Lopez, Begona; Hermida, Nerea; Gonzalez, Arantxa; Dotor, Javier; Borras-Cuesta, Francisco; Diez, Javier; Fortuno, Ana; Zalba, Guillermo

2012-01-01

121

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

2013-02-28

122

?2 adrenergic receptor activation induces microglial NADPH oxidase activation and dopaminergic neurotoxicity through an ERK-dependent/Protein Kinase A-independent pathway  

PubMed Central

Activation of the ?2 adrenergic receptor (?2AR) on immune cells has been reported to possess anti-inflammatory properties, however, the pro-inflammatory properties of ?2AR activation remain unclear. In this study, using rat primary mesencephalic neuron-glia cultures, we report that salmeterol, a long-acting ?2AR agonist, selectively induces dopaminergic (DA) neurotoxicity through its ability to activate microglia. Salmeterol selectively increased the production of reactive oxygen species (ROS) by NADPH oxidase (PHOX), the superoxide-producing enzyme in microglia. A key role of PHOX in mediating salmeterol-induced neurotoxicity was demonstrated by the inhibition of DA neurotoxicity in cultures pretreated with diphenylene-iodonium (DPI), an inhibitor of PHOX activity. Mechanistic studies revealed the activation of microglia by salmeterol results in the selective phosphorylation of ERK, a signaling pathway required for the translocation of the PHOX cytosolic subunit p47phox to the cell membrane. Furthermore, we found ERK inhibition, but not protein kinase A (PKA) inhibition, significantly abolished salmeterol-induced superoxide production, p47phox translocation, and its ability to mediate neurotoxicity. Together, these findings indicate that ?2AR activation induces microglial PHOX activation and DA neurotoxicity through an ERK-dependent/PKA-independent pathway.

Qian, Li; Hu, Xiaoming; Zhang, Dan; Snyder, Amanda; Wu, Hung-ming; Li, Yachen; Wilson, Belinda; Lu, Ru-Bin; Hong, Jau-Shyong; Flood, Patrick M

2013-01-01

123

Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries: regulation by angiotensin II.  

PubMed

A major source of vascular smooth muscle cell (VSMC) superoxide is NAD(P)H oxidase. However, the molecular characteristics and regulation of this enzyme are unclear. We investigated whether VSMCs from human resistance arteries (HVSMCs) possess a functionally active, angiotensin II (Ang II)-regulated NAD(P)H oxidase that contains neutrophil oxidase subunits, including p22phox, gp91phox, p40phox, p47phox, and p67phox. mRNA expression of gp91phox homologues, nox1 and nox4, was also assessed in HVSMCs, human aortic smooth muscle cells, and rat VSMCs. HVSMCs were obtained from resistance arteries from gluteal biopsies of healthy subjects. gp91phox and nox4, but not nox1, were detected in HVSMCs. Nox1 and nox4, but not gp91phox, were expressed in human aortic smooth muscle cells and rat VSMCs. All NAD(P)H oxidase subunits were present in HVSMCs as detected by reverse transcriptase-polymerase chain reaction and immunoblotting. Ang II increased NAD(P)H oxidase subunit abundance. These effects were inhibited by cycloheximide. Acute Ang II stimulation (10 to 15 minutes) increased p47phox serine phosphorylation and induced p47phox and p67phox translocation. This was associated with NAD(P)H oxidase activation. In cells transfected with gp91phox antisense oligonucleotides, Ang II-mediated actions were abrogated. NADPH-induced superoxide generation was reduced by gp91ds-tat and apocynin, inhibitors of p47phox-gp91phox interactions. Our results suggest that HVSMCs possess a functionally active gp91phox-containing neutrophil-like NAD(P)H oxidase. Ang II regulates the enzyme by inducing phosphorylation of p47phox, translocation of cytosolic subunits, and de novo protein synthesis. These novel findings provide insight into the molecular regulation of NAD(P)H oxidase by Ang II in HVSMCs. Furthermore, we identify differences in gp91phox homologue expression in VSMCs from rats and human small and large arteries. PMID:12065324

Touyz, Rhian M; Chen, Xin; Tabet, Fatiha; Yao, Guoying; He, Gang; Quinn, Mark T; Pagano, Patrick J; Schiffrin, Ernesto L

2002-06-14

124

NADPH oxidase 4 regulates cardiomyocyte differentiation via redox activation of c-Jun protein and the cis-regulation of GATA-4 gene transcription.  

PubMed

NADPH oxidase 4 (Nox4) generates reactive oxygen species (ROS) that can modulate cellular phenotype and function in part through the redox modulation of the activity of transcription factors. We demonstrate here the potential of Nox4 to drive cardiomyocyte differentiation in pluripotent embryonal carcinoma cells, and we show that this involves the redox activation of c-Jun. This in turn acts to up-regulate GATA-4 expression, one of the earliest markers of cardiotypic differentiation, through a defined and highly conserved cis-acting motif within the GATA-4 promoter. These data therefore suggest a mechanism whereby ROS act in pluripotential cells in vivo to regulate the initial transcription of critical tissue-restricted determinant(s) of the cardiomyocyte phenotype, including GATA-4. The ROS-dependent activation, mediated by Nox4, of widely expressed redox-regulated transcription factors, such as c-Jun, is fundamental to this process. PMID:23589292

Murray, Thomas V A; Smyrnias, Ioannis; Shah, Ajay M; Brewer, Alison C

2013-04-15

125

Heme oxygenase-1 regulates matrix metalloproteinase MMP-1 secretion and chondrocyte cell death via Nox4 NADPH oxidase activity in chondrocytes.  

PubMed

Interleukin-1? (IL-1?) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1? stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1? signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22(phox) heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1?. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis. PMID:23840483

Rousset, Francis; Nguyen, Minh Vu Chuong; Grange, Laurent; Morel, Françoise; Lardy, Bernard

2013-06-20

126

Activity of a purified nonsteroidal gametic factor on the inducibility of hepatic delta-aminolevulinic acid synthetase, NADPH-oxidase and tyrosine aminotransferase in senescent rats.  

PubMed

The effect of a water soluble nonsteroidal factor extracted from the male gamete on the activity of certain liver inducible enzymes during aging has been examined. Three enzymes have been studied: delta-aminolevulinic acid synthetase, NADPH-oxidase and tyrosine aminotransferase whose inducibility by ethanol, phenobarbital and ACTH, respectively, show age dependent alterations. The results here reported show that this factor is able to restore the enzyme inducibility in the liver of aging (600-day-old) rats without affecting the response of young (40-day-old) rats. Since the enzyme inducibility is altered during aging, and in the majority of rat hepatomas this factor might enter, possibly, in the regulation of enzyme activity also of neoplastic cells. PMID:6108799

Casellato, M M; Manera, E; Riboni, L; Comolli, R; Lugaro, G

1980-01-01

127

A novel post-translational incorporation of tyrosine into multiple proteins in activated human neutrophils. Correlation with phagocytosis and activation of the NADPH oxidase-mediated respiratory burst.  

PubMed

Activation of human neutrophils by PMA causes a post-translational incorporation of 14C-labeled tyrosine into multiple neutrophil (PMN) proteins, that is distinctly different from the enzymatic tyrosinolation of tubulin in FMLP-stimulated PMN. Post-translational incorporation of other radiolabeled amino acids, including the structurally similar amino acid phenylalanine, does not occur under identical conditions of neutrophil activation, suggesting an involvement of the phenolic hydroxyl group of tyrosine in the PMA-mediated reaction. Similar to the stimulation of PMN tubulin tyrosinolation by FMLP, the PMA-induced incorporation of tyrosine into multiple PMN proteins is closely associated with activation of the NADPH oxidase-mediated respiratory burst in stimulated PMN and can be inhibited by a variety of reducing agents, inhibitors of peroxidase-mediated reactions, and intracellular scavengers of oxygen radicals. Moreover, the PMA-induced post-translational incorporation of tyrosine does not occur in PMN from patients with chronic granulomatous disease and is significantly reduced (50%) in PMN of an individual with myeloperoxidase deficiency. A similar stimulus-induced incorporation of tyrosine into multiple PMN proteins is also observed in PMN exposed to various phagocytic stimuli, and the incorporated radioactivity in cells undergoing phagocytosis is substantially enriched (40- to 50-fold) in isolated PMN phagolysosomes. Consistent with this latter observation, HPLC fractionation of stimulated PMN proteins and analysis of the incorporated radioactivity reveal that the 14C label is primarily associated with PMN membrane proteins. Furthermore, this post-translational incorporation of tyrosine, like that associated with PMA stimulation, is associated with production of oxygen radicals and the generation of protein carbonyl derivatives, which are indicative of oxidative protein modifications via mixed function oxidases. Our findings indicate that tyrosine incorporation into membrane proteins of stimulated PMN is functionally relevant to the physiologic host-defense responses of human neutrophils undergoing phagocytosis. PMID:1331234

Nath, J; Ohno, Y; Gallin, J I; Wright, D G

1992-11-15

128

Flow increases superoxide production by NADPH oxidase via activation of Na-K-2Cl cotransport and mechanical stress in thick ascending limbs.  

PubMed

Superoxide (O(2)(-)) regulates renal function and is implicated in hypertension. O(2)(-) production increases in response to increased ion delivery in thick ascending limbs (TALs) and macula densa and mechanical strain in other cell types. Tubular flow in the kidney acutely varies causing changes in ion delivery and mechanical stress. We hypothesized that increasing luminal flow stimulates O(2)(-) production by NADPH oxidase in TALs via activation of Na-K-2Cl cotransport. We measured intracellular O(2)(-) in isolated rat TALs using dihydroethidium in the presence and absence of luminal flow and inhibitors of NADPH oxidase, Na-K-2Cl cotransport, and Na/H exchange. In the absence of flow, the rate of O(2)(-) production was 5.8 +/- 1.4 AU/s. After flow was initiated, it increased to 29.7 +/- 4.3 AU/s (P < 0.001). O(2)(-) production was linearly related to flow. Tempol alone and apocynin alone blocked the flow-induced increase in O(2)(-) production (3.5 +/- 1.7 vs. 4.5 +/- 2.8 AU/s and 8.2 +/- 2.1 vs. 10.6 +/- 2.8 AU/s, respectively). Furosemide decreased flow-induced O(2)(-) production by 55% (37.3 +/- 5.2 to 16.8 +/- 2.8 AU/s; P < 0.002); however, dimethylamiloride had no effect. Finally, we examined whether changes in mechanical forces are involved in flow-induced O(2)(-) production by using a Na-free solution to perfuse TALs. In the absence of NaCl, luminal flow enhanced O(2)(-) production (1.5 +/- 0.5 to 13.5 +/- 1.1 AU/s; P < 0.001), approximately 50% less stimulation than when flow was increased in the presence of luminal NaCl. We conclude that flow stimulates O(2)(-) production in TALs via activation of NADPH oxidase and that NaCl absorption due to Na-K-2Cl cotransport and flow-associated mechanical factors contribute equally to this process. PMID:17132867

Hong, Nancy J; Garvin, Jeffrey L

2006-11-28

129

Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Characterization of the membrane-associated component.  

PubMed

Sodium dodecyl sulfate (SDS) was shown to elicit NADPH-dependent superoxide (O2-) production by a cell-free system derived from sonically disrupted resting guinea pig macrophages (Bromberg, Y., and Pick, E. (1985) J. Biol. Chem. 260, 13539-13545). O2- production was absolutely dependent on the cooperation between a membrane-associated component, sedimenting with the 48,000 X g pellet and a cytosolic factor, nonsedimentable at 265,000 X g. The present report describes the solubilization and characterization of the membrane-associated component of the SDS-activable O2(-)-forming NADPH oxidase (operationally termed pi). Treatment of the 48,000 X g pellet with 30 mM octyl glucoside resulted in complete transfer of pi to the soluble fraction. The solubilized pellet produced an average of 0.92 mumol of O2-/mg of protein/min upon reduction of octyl glucoside content below the critical micellar concentration and in the presence of cytosol, 100 microM SDS, and 0.2 mM NADPH. The activity of solubilized pellet-cytosol combinations was also expressed as NADPH-dependent, azide-resistant oxygen consumption and hydrogen peroxide production. pi was inactivated by the sulfhydryl reagent p-chloromercuribenzoate. Solubilized pellet contained spectroscopically detectable cytochrome b559 (225.6 +/- 15.0 pmol/mg mg protein). Both pi and cytochrome b559 were bound by Cibacron Blue Sepharose and could be eluted by a gradient of octyl glucoside (0-30 mM) in the presence of 1 M KCl. On high performance gel filtration on Superose 12, both pi and cytochrome b559 eluted in the excluded volume; when 25 mM octyl glucoside was present in the elution buffer, pi was partially dissociated from cytochrome b559. Sequential purification of pi on Blue Sepharose followed by gel filtration on Superose 12 in the presence of 25 mM octyl glucoside lead to complete resolution of pi from cytochrome b559 (pi was found in the Mr = 28,000 - 11,000 range while the bulk of cytochrome b559 eluted in the Mr = 113,000 - 71,000 range). We propose that pi is distinct from cytochrome b559 and represents a membrane-associated component in an amphiphile-activated electron transport chain from NADPH to oxygen. PMID:2824496

Pick, E; Bromberg, Y; Shpungin, S; Gadba, R

1987-12-01

130

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.

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

2010-01-01

131

Tobacco Nectaries Express a Novel NADPH Oxidase Implicated in the Defense of Floral Reproductive Tissues against Microorganisms1[OA  

PubMed Central

Hydrogen peroxide produced from the nectar redox cycle was shown to be a major factor contributing to inhibition of most microbial growth in floral nectar; however, this obstacle can be overcome by the floral pathogen Erwinia amylovora. To identify the source of superoxide that leads to hydrogen peroxide accumulation in nectary tissues, nectaries were stained with nitroblue tetrazolium. Superoxide production was localized near nectary pores and inhibited by diphenylene iodonium but not by cyanide or azide, suggesting that NAD(P)H oxidase is the source of superoxide. Native PAGE assays demonstrated that NADPH (not NADH) was capable of driving the production of superoxide, diphenyleneiodonium chloride was an efficient inhibitor of this activity, but cyanide and azide did not inhibit. These results confirm that the production of superoxide was due to an NADPH oxidase. The nectary enzyme complex was distinct by migration on gels from the leaf enzyme complex. Temporal expression patterns demonstrated that the superoxide production (NADPH oxidase activity) was coordinated with nectar secretion, the expression of Nectarin I (a superoxide dismutase in nectar), and the expression of NOX1, a putative gene for a nectary NADPH oxidase that was cloned from nectaries and identified as an rbohD-like NADPH oxidase. Further, in situ hybridization studies indicated that the NADPH oxidase was expressed in the early stages of flower development although superoxide was generated at later stages (after Stage 10), implicating posttranslational regulation of the NADPH oxidase in the nectary.

Carter, Clay; Healy, Rosanne; O'Tool, Nicole M.; Naqvi, S.M. Saqlan; Ren, Gang; Park, Sanggyu; Beattie, Gwyn A.; Horner, Harry T.; Thornburg, Robert W.

2007-01-01

132

Bile Acid Reflux Contributes To Development of Esophageal Adenocarcinoma Via Activation Of Phosphatidylinositol-Specific Phospholipase C?2 And NADPH Oxidase NOX5-S  

PubMed Central

Gastroesophageal reflux disease complicated by Barrett’s esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid-induced in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, H2O2 production and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibiter of PI-PLC. PI-PLC?1, ?3, ?4, ?1 and ?2, but not ?2 and ?1 were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLC?2 or ERK-2 MAP kinase with siRNAs significantly decreased TDCA-induced increase in NOX5-S expression, H2O2 production and cell proliferation. In contrast, knockdown of PI-PLC ?1, ?3, ?4, ?1 or ERK-1 MAP kinase had no significant effect. TDCA significantly increased ERK-2 phosphorylation, an increase which was reduced by U73122 or PI-PLC?2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLC?2 and ERK-2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with Barrett’s esophagus may increase ROS production and cell proliferation via activation of PI-PLC?2, ERK-2 MAP kinase and NADPH oxidase NOX5-S, thereby contributing to the development of EA.

Hong, Jie; Behar, Jose; Wands, Jack; Resnick, Murray; Wang, Li Juan; DeLellis, Ronald A.; Lambeth, David; Cao, Weibiao

2009-01-01

133

NADPH Oxidase Is Internalized by Clathrin-coated Pits and Localizes to a Rab27A/B GTPase-regulated Secretory Compartment in Activated Macrophages*  

PubMed Central

Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b558) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91phox and CeCl3 cytochemistry showed the presence of gp91phox and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b558 is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b558-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5?-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b558 under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNF?, and CD40L as physiological inducers of cyt b558 exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b558, which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b558 did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b558 depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell.

Ejlerskov, Patrick; Christensen, Dan Ploug; Beyaie, David; Burritt, James B.; Paclet, Marie-Helene; Gorlach, Agnes; van Deurs, Bo; Vilhardt, Frederik

2012-01-01

134

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation  

PubMed Central

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.

Pescatore, Luciana A.; Bonatto, Diego; Forti, Fabio L.; Sadok, Amine; Kovacic, Herve; Laurindo, Francisco R. M.

2012-01-01

135

Rosuvastatin blocks advanced glycation end products-elicited reduction of macrophage cholesterol efflux by suppressing NADPH oxidase activity via inhibition of geranylgeranylation of Rac-1.  

PubMed

Adenosine triphosphate-binding membrane cassette transporter A1 (ABCA1) and ABCG1 play a crucial role in macrophage cholesterol efflux, which is a novel therapeutic target for atherosclerosis. Advanced glycation end products (AGE) and their receptor RAGE axis is involved in accelerated atherosclerosis in diabetes as well. However, the role of AGE-RAGE axis in macrophage cholesterol efflux is not fully understood. We examined here whether AGE-RAGE axis could impair cholesterol efflux from human macrophage cells, THP-1 cells by suppressing ABCA1 and ABCG1 expression. We further investigated the effects of rosuvastatin on cholesterol efflux from AGE-exposed THP-1 cells. AGE increased reactive oxygen species generation in THP-1 cells, which was completely inhibited by rosuvastatin, anti-RAGE-antibody or diphenylene iodonium chloride (DPI), an inhibitor of NADPH oxidase. The antioxidative effect of rosuvastatin on AGE-exposed THP-1 cells was significantly prevented by geranylgeranyl pyrophosphate (GGPP). AGE decreased ABCA1 and ABCG1 mRNA levels, and subsequently reduced cholesterol efflux from THP-1 cells, which was prevented by GGPP. DPI mimicked the effects of rosuvastain. The results demonstrated that rosuvastatin could inhibit the AGE-induced reduction of THP-1 macrophage cholesterol efflux by suppressing NADPH oxidase activity via inhibition of geranylgeranylation of Rac-1. Our present study provides a novel beneficial aspect of rosuvastatin in diabetes; rosuvastain may prevent the development and progression of atherosclerosis in diabetes by not only reducing serum cholesterol level, but also by improving cholesterol efflux from foam cells of the arterial wall via blocking the harmful effects of AGE on macrophages. PMID:21823057

Ishibashi, Y; Matsui, T; Takeuchi, M; Yamagishi, S

2011-08-05

136

Marinobufagenin and cyclic strain may activate endothelial NADPH oxidase, contributing to the adverse impact of salty diets on vascular and cerebral health.  

PubMed

Limited but provocative ecologic epidemiology suggests that dietary salt may play a central role in the genesis of not only of stroke, but also dementia, including Alzheimer's disease. Impairment of nitric oxide bioactivity in the cerebral microvasculature is a likely mediator of this effect. Salted diets evoke increased adrenal secretion of the natriuretic steroid marinobufagenin (MBG), which promotes natriuresis via inhibition of renal tubular Na+/K+-ATPase; this effect is notably robust in salt-sensitive rodent strains in which other compensatory natriuretic mechanisms are subnormally efficient. MBG-mediated inhibition of sodium pumps in vascular smooth muscle likely plays a role in the hypertension induced by salty diets in these rodents. However, salt sensitivity in humans is associated with increased vascular mortality and ventricular hypertrophy independent of blood pressure; this suggests that MBG may be pathogenic via mechanisms unrelated to blood pressure control. Indeed, recent evidence indicates that MBG, via interaction with alpha1 isoforms of the sodium pump, can activate various intracellular signaling pathways at physiological concentrations too low to notably inhibit pump activity. An overview of current evidence suggests the hypothesis that MBG - as well as the cyclic strain induced by hypertension per se - may induce endothelial oxidative stress by activating NADPH oxidase. If so, this could rationalize the increase in vascular and systemic oxidative stress observed in salt-sensitive rodents fed salty diets, or in rodents infused with MBG; moreover, if this effect is a particularly prominent determinant of oxidative stress in cerebrovascular endothelium, it might help to explain the virtual absence of stroke and dementia in low-salt societies. As a corollary of this hypothesis, it can be predicted that spirulina-derived phycobilins, which appear to mimic the physiological role of bilirubin as an inhibitor of NAPDH oxidase complexes, may have potential for ameliorating the adverse health impacts of MBG and of salty diets. Potassium-rich diets are also likely to be protective in this regard, as they should suppress MBG production via their natriuretic impact, while their stimulatory effect on sodium pump activity may exert a hyperpolarizing effect on plasma membranes that suppresses NADPH oxidase activity. PMID:21968275

McCarty, Mark F

2011-10-02

137

Expression and Cellular Localization of Classic NADPH Oxidase Subunits in the Spontaneously Hypertensive Rat Kidney  

Microsoft Academic Search

Phagocytes generate superoxide anion (O2) by a classic, 5-component NADPH oxidase. O2 contributes to hypertension in spontaneously hypertensive rats (SHR). Therefore, we tested the hypothesis that NADPH oxidase expression is enhanced in the SHR kidney. We also analyzed the localization of NADPH oxidase components in SHR kidney. Renal NADPH oxidase was quantified by reverse transcription-polymerase chain reaction and Western blotting

Tinatin Chabrashvili; Akahiro Tojo; Maristela Lika Onozato; Chagriya Kitiyakara; Mark T. Quinn; Toshiro Fujita; William J. Welch; Christopher S. Wilcox

2010-01-01

138

NADPH oxidase-dependent signaling in endothelial cells: role in physiology and pathophysiology.  

PubMed

Reactive oxygen species (ROS) including superoxide (O(2)(.-)) and hydrogen peroxide (H(2)O(2)) are produced endogenously in response to cytokines, growth factors; G-protein coupled receptors, and shear stress in endothelial cells (ECs). ROS function as signaling molecules to mediate various biological responses such as gene expression, cell proliferation, migration, angiogenesis, apoptosis, and senescence in ECs. Signal transduction activated by ROS, "oxidant signaling," has received intense investigation. Excess amount of ROS contribute to various pathophysiologies, including endothelial dysfunction, atherosclerosis, hypertension, diabetes, and acute respiratory distress syndrome (ARDS). The major source of ROS in EC is a NADPH oxidase. The prototype phagaocytic NADPH oxidase is composed of membrane-bound gp91phox and p22hox, as well as cytosolic subunits such as p47(phox), p67(phox) and small GTPase Rac. In ECs, in addition to all the components of phagocytic NADPH oxidases, homologues of gp91(phox) (Nox2) including Nox1, Nox4, and Nox5 are expressed. The aim of this review is to provide an overview of the emerging area of ROS derived from NADPH oxidase and oxidant signaling in ECs linked to physiological and pathophysiological functions. Understanding these mechanisms may provide insight into the NADPH oxidase and oxidant signaling components as potential therapeutic targets. PMID:18783313

Frey, Randall S; Ushio-Fukai, Masuko; Malik, Asrar B

2009-04-01

139

NADPH Oxidase-Dependent Signaling in Endothelial Cells: Role in Physiology and Pathophysiology  

PubMed Central

Abstract Reactive oxygen species (ROS) including superoxide (O2·?) and hydrogen peroxide (H2O2) are produced endogenously in response to cytokines, growth factors; G-protein coupled receptors, and shear stress in endothelial cells (ECs). ROS function as signaling molecules to mediate various biological responses such as gene expression, cell proliferation, migration, angiogenesis, apoptosis, and senescence in ECs. Signal transduction activated by ROS, “oxidant signaling,” has received intense investigation. Excess amount of ROS contribute to various pathophysiologies, including endothelial dysfunction, atherosclerosis, hypertension, diabetes, and acute respiratory distress syndrome (ARDS). The major source of ROS in EC is a NADPH oxidase. The prototype phagaocytic NADPH oxidase is composed of membrane-bound gp91phox and p22hox, as well as cytosolic subunits such as p47phox, p67phox and small GTPase Rac. In ECs, in addition to all the components of phagocytic NADPH oxidases, homologues of gp91phox (Nox2) including Nox1, Nox4, and Nox5 are expressed. The aim of this review is to provide an overview of the emerging area of ROS derived from NADPH oxidase and oxidant signaling in ECs linked to physiological and pathophysiological functions. Understanding these mechanisms may provide insight into the NADPH oxidase and oxidant signaling components as potential therapeutic targets. Antioxid. Redox Signal. 11, 791–810.

Ushio-Fukai, Masuko; Malik, Asrar B.

2009-01-01

140

Localization of NADPH Oxidase in Sympathetic and Sensory Ganglion Neurons and Perivascular Nerve Fibers  

PubMed Central

Superoxide anion (O2?•) production was previously reported to be increased in celiac ganglia (CG) during DOCA-salt hypertension, possibly via activation of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. This suggested a role for neuronal NADPH oxidase in autonomic neurovascular control. However, the expression and localization of NADPH oxidase in the peripheral neurons is not fully known. The purpose of this study was to examine the subcellular localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers. In rat CG, p22phox and neuropeptide Y (NPY) were colocalized in all neurons. P22phox was also localized to dorsal root ganglia (DRG) neurons that contain calcitonin gene related peptide (CGRP). In mesenteric arteries, p22phox and p47phox were colocalized with NPY or CGRP in perivascular nerve terminals. A similar pattern of nerve terminal staining of p22phox and p47phox was also found in cultured CG neurons and nerve growth factor (NGF)-differentiated PC12 cells. These data demonstrate a previously uncharacterized localization of NADPH oxidase in perivascular nerve fibers. The presence of a O2?• – generating enzyme in close vicinity to the sites of neurotransmitter handling in the nerve fibers suggests the possibility of novel redox-mediated mechanisms in peripheral neurovascular control.

Cao, Xian; Demel, Stacie L.; Quinn, Mark T.; Galligan, James J.; Kreulen, David L.

2009-01-01

141

Physiological roles of NOX/NADPH oxidase, the superoxide-generating enzyme  

PubMed Central

NADPH oxidase is a superoxide (O2•?)-generating enzyme first identified in phagocytes, essential for their bactericidal activities. Later, in non-phagocytes, production of O2•? was also demonstrated in an NADPH-dependent manner. In the last decade, several non-phagocyte-type NADPH oxidases have been identified. The catalytic subunit of these oxidases, NOX, constitutes the NOX family. There are five homologs in the family, NOX1 to NOX5, and two related enzymes, DUOX1 and DUOX2. Transgenic or gene-disrupted mice of the NOX family have also been established. NOX/DUOX proteins possess distinct features in the dependency on other components for their enzymatic activities, tissue distributions, and physiological functions. This review summarized the characteristics of the NOX family proteins, especially focused on their functions clarified through studies using gene-modified mice.

Katsuyama, Masato; Matsuno, Kuniharu; Yabe-Nishimura, Chihiro

2012-01-01

142

Mechanisms of inflammatory neurodegeneration: iNOS and NADPH oxidase.  

PubMed

Inflammation contributes to a wide variety of brain pathologies, apparently via glia killing neurons. A number of mechanisms by which inflammatory-activated microglia and astrocytes kill neurons have been identified in culture. These include iNOS (inducible nitric oxide synthase), which is expressed in glia only during inflammation, and PHOX (phagocytic NADPH oxidase) found in microglia and acutely activated by inflammation. High levels of iNOS expression in glia cause (i) NO (nitric oxide) inhibition of neuronal respiration, resulting in neuronal depolarization and glutamate release, followed by excitotoxicity, and (ii) glutamate release from astrocytes via calcium-dependent vesicular release. Hypoxia strongly synergizes with iNOS expression to induce neuronal death via mechanism (i), because NO inhibits cytochrome oxidase in competition with oxygen. Activation of PHOX (by cytokines, beta-amyloid, prion protein, ATP or arachidonate) causes microglial proliferation and inflammatory activation; thus PHOX is a key regulator of inflammation. Activation of PHOX alone causes no death, but when combined with expressed iNOS results in extensive neuronal death via peroxynitrite production. PMID:17956292

Brown, G C

2007-11-01

143

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. J. Cell. Physiol. 229: 100-107, 2014. © 2013 Wiley Periodicals, Inc. PMID:23804302

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

2014-01-01

144

Regulation of the NADPH oxidase and associated ion fluxes during phagocytosis.  

PubMed

The production of reactive oxygen species (ROS) within immune cell phagosomes is critical for antimicrobial activity and for correct antigen processing, and influences signaling pathways that direct host responses to infection and inflammation. Because excess oxidants can cause tissue damage and oxidative stress, phagocytes must precisely control both the location and timing of NADPH oxidase activity. How differential regulation is achieved at phagosomes is not well understood. Recent studies have revealed that the PI(3)P phosphoinositide plays an important role in locally boosting phagosomal NADPH oxidase activity through its binding to the p40(phox) NADPH oxidase subunit. Furthermore, phox subunit dynamics at phagosomes may regulate the timing of the oxidative burst. Novel elements regulating catalytic core trafficking include Rab27 and SNAP-23. In addition to trafficking events, the activity of the electrogenic oxidase is also governed by ionic fluxes, which are constrained at phagosomes owing to low intraphagosomal volume and dynamic display of channels, transporters, and pumps. New insights on the interdependence of phagosomal pH and ROS have been recently elucidated, and chloride channels important for microbicidal functions, including CFTR, and CLIC family channels, have been identified. Finally, periphagosomal calcium microdomains and calcium-dependent S100A8/9 protein recruitment may help fine-tune spatiotemporal regulation of NADPH oxidase activation for an effective immune response. PMID:23980663

Nunes, Paula; Demaurex, Nicolas; Dinauer, Mary C

2013-09-16

145

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

146

Involvement of NADPH Oxidases in Cardiac Remodelling and Heart Failure  

Microsoft Academic Search

Cardiac remodelling occurs in response to stress, such as chronic hypertension or myocardial infarction, and forms the substrate for subsequent development of heart failure. Key pathophysiological features include ventricular hypertrophy, interstitial fibrosis, contractile dysfunction, and chamber dilatation. Although the molecular mechanisms are complex and not fully defined, substantial evidence now implicates increased oxidative stress as being important. The NADPH oxidase

Alexander Sirker; Min Zhang; Colin Murdoch; Ajay M. Shah

2007-01-01

147

A central role for the endothelial NADPH oxidase in atherosclerosis  

Microsoft Academic Search

An increasing body of evidence has demonstrated that NADPH oxidase plays a critical role in several early steps leading toward the development of atherosclerosis. These effects appear to be carried out by both the ability of O2? to act as a small second messenger molecule, and potentially the oxidation of low density lipoprotein by O2?. We describe a model for

Jamie W. Meyer; Mark E. Schmitt

2000-01-01

148

Characterisation of electron currents generated by the human neutrophil NADPH oxidase  

SciTech Connect

Electron transport by the human neutrophil NADPH oxidase is an important microbicidal weapon for phagocytes. The electron current (I{sub e}) generated by the neutrophil NADPH oxidase is poorly characterised due to the lack of appropriate electrophysiological data. In this study, I fully characterise the neutrophil generated I{sub e} when the NADPH oxidase is activated by NADPH and GTP{gamma}S. The neutrophil I{sub e} was markedly voltage-dependent in the entire voltage range in comparison to those electron currents measured after chloride was removed from the external bath solution. The difference in I{sub e} measured in chloride free conditions was not due to a change in the activation kinetics of voltage-gated proton channels. The I{sub e} depolarises the neutrophil plasma membrane at a rate of 2.3 V s{sup -1} and this depolarisation was opposed when voltage-gated proton channels are activated. 3 mM ZnCl{sub 2} depolarised the membrane potential to +97.8 {+-} 2.5 mV (n = 4), and this depolarisation was abolished after NADPH oxidase inhibition.

Ahluwalia, Jatinder [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom)], E-mail: j.ahluwalia@uel.ac.uk

2008-04-11

149

Fungal Gliotoxin Targets the Onset of Superoxide-Generating NADPH Oxidase of Human Neutrophils  

Microsoft Academic Search

Gliotoxin from Aspergillus, bearing a S?S bond in its structure, prevented the onset of O?2 generation by the human neutrophil NADPH oxidase in response to phorbol myristate acetate (PMA). Gliotoxin affected the activation process harder than the activated oxidase, as shown by its stronger inhibition when added to neutrophils prior to, than post-PMA at maximum enzyme turnover. Decreased O?2 generation

Lucia S. Yoshida; Shigeru Abe; Shohko Tsunawaki

2000-01-01

150

Nox4 as the Major Catalytic Component of an Endothelial NAD(P)H Oxidase  

Microsoft Academic Search

Background—Recent evidence has suggested that reactive oxygen species are important signaling molecules in vascular cells and play a pivotal role in the development of vascular diseases. The activity of NAD(P)H oxidase has been identified as the major source of reactive oxygen species in vascular endothelial cells. However, the precise molecular structure and the mechanism of activation of the oxidase have

Tetsuro Ago; Takanari Kitazono; Hiroaki Ooboshi; Teruaki Iyama; Youn Hee Han; Junichi Takada; Masanori Wakisaka; Setsuro Ibayashi; Hideo Utsumi; Mitsuo Iida

2010-01-01

151

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

PubMed

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 activating Nrf2, a transcriptional factor that induces antioxidative 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 mice and Nrf2(-/-) mice compared with 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 proinflammatory and injurious product of cellular necrosis, in cell-free bronchoalveolar lavage fluid. Pharmacologic activation of Nrf2 by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl]imidazole 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

2013-01-07

152

NOX3 NADPH Oxidase Couples Transient Receptor Potential Vanilloid 1 to Signal Transducer and Activator of Transcription 1-Mediated Inflammation and Hearing Loss  

PubMed Central

Abstract Transient receptor potential vanilloid 1 (TRPV1) is implicated in cisplatin ototoxicity. Activation of this channel by cisplatin increases reactive oxygen species generation, which contribute to loss of outer hair cells in the cochlea. Knockdown of TRPV1 by short interfering RNA protected against cisplatin ototoxicity. In this study, we examined the mechanism underlying TRPV1-mediated ototoxicity using cultured organ of Corti transformed cells (UB/OC-1) and rats. Trans-tympanic injections of capsaicin produced transient hearing loss within 24?h, which recovered by 72?h. In UB/OC-1 cells, capsaicin increased NOX3 NADPH oxidase activity and activation of signal transducer and activator of transcription 1 (STAT1). Intratympanic administration of capsaicin transiently increased STAT1 activity and expression of downstream proinflammatory molecules. Capsaicin produced a transient increase in CD14-positive inflammatory cells into the cochlea, which mimicked the temporal course of STAT1 activation but did not alter the expression of apoptotic genes or damage to outer hair cells. In addition, trans-tympanic administration of STAT1 short interfering RNA protected against capsaicin-induced hearing loss. These data suggest that activation of TRPV1 mediates temporary hearing loss by initiating an inflammatory process in the cochlea via activation of NOX3 and STAT1. Thus, these proteins represent reasonable targets for ameliorating hearing loss. Antioxid. Redox Signal. 14, 999–1010.

Mukherjea, Debashree; Jajoo, Sarvesh; Sheehan, Kelly; Kaur, Tejbeer; Sheth, Sandeep; Bunch, Jennifer; Perro, Christopher; Rybak, Leonard P.

2011-01-01

153

Cross-talk between IRAK-4 and the NADPH oxidase1  

PubMed Central

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

Pacquelet, Sandrine; Johnson, Jennifer L.; Ellis, Beverly A.; Brzezinska, Agnieszka A.; Lane, William S.; Munafo, Daniela B.; Catz, Sergio D.

2007-01-01

154

Cellular crosstalk between TNF-?, NADPH oxidase, PKC?2, and C2GNT in human leukocytes.  

PubMed

Increasing evidence suggests that chronic, sub-clinical inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have established the potential role of the inflammatory enzyme, core 2 ?-1, 6-N-acetylglucosaminyltransferase (C2GNT) in diabetic retinopathy. The present study was designed to explore the NADPH oxidase signaling pathway in the tumor necrosis factor-alpha (TNF-?)-induced activity of C2GNT in leukocytes. Human leukocytes (U937 cells) and an Epstein-Barr-transformed lymphoblastoid cell line deficient in p47phox (F10007 cells) were used for the study. Cells were exposed to TNF-? for 24h in the presence and absence of 1) NADPH oxidase inhibitors (apocynin and scrambled and unscrambled gp91ds-tat), 2) LY379196 (specific protein kinase C ?1/2 (PKC?1/2) inhibitor), and 3) the antioxidant tiron. Subsequent C2GNT and NADPH activity was measured and the adhesion of U937 and F10007 cells to endothelial cells was assessed. TNF-?-induced C2GNT activity (1813±326 pmol/h/mg protein) (mean±SEM) in human leukocytes was significantly reversed with apocynin (153±82 pmol/h/mg protein), unscrambled gp91ds-tat (244±122 pmol/h/mg protein) and tiron (756±87 pmol/h/mg protein). We further supported this C2GNT-NADPH oxidase link using p47phox-deficient leukocytes. The deficiency in p47phox prevented TNF-?-induced NADPH oxidase and C2GNT activity and adherence to endothelial cells. The response to TNF-? was restored by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. Our results demonstrate for the first time a novel signaling crosstalk between TNF-?, NADPH oxidase, PKC?1/2 and C2GNT in leukocytes. PMID:22182514

Tarr, Joanna M; Ding, Ning; Kaul, Kirti; Antonell, Anna; Pérez-Jurado, Luis A; Chibber, Rakesh

2011-12-13

155

NADPH oxidases: functions and pathologies in the vasculature.  

PubMed

Reactive oxygen species are ubiquitous signaling molecules in biological systems. Four members of the NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species in the vasculature: Nox1, Nox2, Nox4, and Nox5. Signaling cascades triggered by stresses, hormones, vasoactive agents, and cytokines control the expression and activity of these enzymes and of their regulatory subunits, among which p22phox, p47phox, Noxa1, and p67phox are present in blood vessels. Vascular Nox enzymes are also regulated by Rac, ClC-3, Poldip2, and protein disulfide isomerase. Multiple Nox subtypes, simultaneously present in different subcellular compartments, produce specific amounts of superoxide, some of which is rapidly converted to hydrogen peroxide. The identity and location of these reactive oxygen species, and of the enzymes that degrade them, determine their downstream signaling pathways. Nox enzymes participate in a broad array of cellular functions, including differentiation, fibrosis, growth, proliferation, apoptosis, cytoskeletal regulation, migration, and contraction. They are involved in vascular pathologies such as hypertension, restenosis, inflammation, atherosclerosis, and diabetes. As our understanding of the regulation of these oxidases progresses, so will our ability to alter their functions and associated pathologies. PMID:19910640

Lassčgue, Bernard; Griendling, Kathy K

2009-11-12

156

Melamine activates NF?B/COX-2/PGE2 pathway and increases NADPH oxidase-dependent ROS production in macrophages and human embryonic kidney cells.  

PubMed

Melamine is a wildly used compound in manufactures of plastics and resins. A variety of toxic effects from melamine, including nephrolithiasis, chronic kidney inflammation, and bladder carcinoma, have been mentioned. Oxidative stress is considered to be an important pathogenic mechanism of kidney disease which may develop from an increasing free radical production through inflammation. The aim of this study is to investigate melamine-induced oxidative stress and inflammation in macrophage-like cell line RAW 264.7 and human embryonic kidney cell line HEK293. Results indicated melamine activated nuclear factor (NF)-?B through increasing I?B-? degradation and NF-?B p65/p50 DNA-binding activity. In addition, melamine significantly increased COX-2 expression and prostaglandin E2 (PGE2) production. Moreover, melamine activated NADPH oxidase (NOX), including NOX1, NOX2 and NOX4, accompanied with an increase in reactive oxygen species (ROS) production. Furthermore, melamine-induced ROS production could be attenuated by apocynin, a NOX inhibitor. In conclusion, our findings suggest melamine increased inflammation and oxidative stress via activation of NF-?B/COX-2 and NOX/ROS pathway, and first revealed the critical role of NOX in melamine-induced ROS production, suggesting the potential of NOX inhibitor against melamine toxicity. PMID:23643631

Kuo, Fu-Chen; Tseng, Yu-Ting; Wu, Sing-Ru; Wu, Ming-Tsang; Lo, Yi-Ching

2013-04-30

157

A Drosophila model for the screening of bioavailable NADPH oxidase inhibitors and antioxidants.  

PubMed

NADPH oxidase is the major source of non-mitochondrial cellular reactive oxygen species (ROS), and also is reported to be a major cause of various diseases including atherosclerosis and hypertension. In order to screen a new curative reagent that can suppress NADPH oxidase activity, we developed a Drosophila melanogaster fly that would overexpress human Dual oxidase 2 (hDuox2), a member of the NADPH oxidase family, as a screening model. These flies (GMR-GAL4/UAS-hDuox2) had a high generation of ROS in the posterior region of the eye discs along with an easily recognizable rough-eye phenotype, which is an ideal and convenient marker for further screening steps. Moreover, the hDuox2-induced rough-eye phenotype can be rescued by feeding with a culture medium containing mulberry leaves (MLs), which reportedly have an antimetabolic effect. Some commercially available antioxidants such as quercetin-3-O-D-glucoside or quercetin-3-O-glucose-6''-acetate, or the naringin contained in MLs and other herbs, also have shown a similar suppressing effect on the rough-eye phenotype. Our results suggest that flavonoid glycoside is absorbed from the intestine and functions in the body of D. melanogaster as it does in mammalian models such as rats. Thus, the GMR-GAL4/UAS-hDuox2 fly line is a promising model for the screening of novel drugs such as NADPH oxidase inhibitors and/or antioxidants. PMID:21312054

Anh, Nguyen Thi Tu; Nishitani, Maiko; Harada, Shigeharu; Yamaguchi, Masamitsu; Kamei, Kaeko

2011-02-11

158

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.

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

2013-01-01

159

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

PubMed

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; Röhm, Marc; Urban, Constantin F; Segal, Brahm H

2013-03-01

160

Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant.  

PubMed

A large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, relied on data obtained with the inhibitor apocynin (4'-hydroxy-3'methoxyacetophenone). Because the mode of action of apocynin, however, is elusive, we determined its mechanism of inhibition on vascular NADPH oxidases. In HEK293 cells overexpressing NADPH oxidase isoforms (Nox1, Nox2, or Nox4), apocynin failed to inhibit superoxide anion generation detected by lucigenin chemiluminescence. In contrast, apocynin interfered with the detection of reactive oxygen species in assay systems selective for hydrogen peroxide or hydroxyl radicals. Importantly, apocynin interfered directly with the detection of peroxides but not superoxide, if generated by xanthine/xanthine oxidase or nonenzymatic systems. In leukocytes, apocynin is a prodrug that is activated by myeloperoxidase, a process that results in the formation of apocynin dimers. Endothelial cells and smooth muscle cells failed to form these dimers and, therefore, are not able to activate apocynin. Dimer formation was, however, observed in Nox-overexpressing HEK293 cells when myeloperoxidase was supplemented. As a consequence, apocynin should only inhibit NADPH oxidase in leukocytes, whereas in vascular cells, the compound could act as an antioxidant. Indeed, in vascular smooth muscle cells, the activation of the redox-sensitive kinases p38-mitogen-activate protein kinase, Akt, and extracellular signal-regulated kinase 1/2 by hydrogen peroxide and by the intracellular radical generator menadione was prevented in the presence of apocynin. These observations indicate that apocynin predominantly acts as an antioxidant in endothelial cells and vascular smooth muscle cells and should not be used as an NADPH oxidase inhibitor in vascular systems. PMID:18086956

Heumüller, Sabine; Wind, Sven; Barbosa-Sicard, Eduardo; Schmidt, Harald H H W; Busse, Rudi; Schröder, Katrin; Brandes, Ralf P

2007-12-17

161

Role of NADPH oxidase in H9c2 cardiac muscle cells exposed to simulated ischaemia-reperfusion.  

PubMed

Oxidative stress is associated with several cardiovascular pathologies, including hypertension, cardiac hypertrophy and heart failure. Although oxidative stress is also increased after ischaemia-reperfusion (I/R), little is known about the role and the activation mechanisms, in cardiac myocytes under these conditions, of NADPH oxidase, a superoxide-producing enzyme. We found that rat cardiac muscle cells (H9c2) subjected to an in vitro simulated ischaemia (substrate-free medium plus hypoxia) followed by 'reperfusion', displayed increased reactive oxygen species (ROS) production attributable to a parallel increase of NADPH oxidase activity. Our investigation on mechanisms responsible for NADPH oxidase activation showed a contribution of both the increase of NOX2 expression and p47(phox) translocation to the membrane. We also found that the increase of NADPH oxidase activity was associated with higher levels of lipid peroxidation, the activation of redox-sensitive kinases, in particular ERK and JNK, and with cell death. Diphenyleneiodonium (DPI), a flavoprotein inhibitor used as NADPH oxidase inhibitor, prevented I/R-induced ROS formation in treated cells, together with the related lipoperoxidative damage, and JNK phosphorylation without affecting ERK activation, resulting in protection against cell death. Our results provide evidence that NADPH oxidase is a key enzyme involved in I/R-induced oxidant generation and suggest it can be a possible target in cardioprotective strategies against I/R injury, a condition of great importance in human pathology. PMID:18754815

Borchi, Elisabetta; Parri, Matteo; Papucci, Laura; Becatti, Matteo; Nassi, Niccolň; Nassi, Paolo; Nediani, Chiara

2009-08-01

162

Molecular composition and regulation of the Nox family NAD(P)H oxidases  

Microsoft Academic Search

Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious by-products in aerobic metabolism with a few exceptions such as their significant role in host defense. The phagocyte NADPH oxidase, dormant in resting cells, becomes activated during phagocytosis to deliberately produce superoxide, a precursor of other microbicidal ROS, thereby playing a crucial role in killing pathogens. The catalytic center of

Hideki Sumimoto; Kei Miyano; Ryu Takeya

2005-01-01

163

Pulsatile Versus Oscillatory Shear Stress Regulates NADPH Oxidase Subunit Expression: Implication for Native LDL Oxidation  

Microsoft Academic Search

Shear stress regulates endothelial nitric oxide and superoxide (O 2 · ) production, implicating the role of NADPH oxidase activity. It is unknown whether shear stress regulates the sources of reactive species production, consequent low-density lipoprotein (LDL) modification, and initiation of inflammatory events. Bovine aortic endothelial cells (BAECs) in the presence of 50 g\\/mL of native LDL were exposed to

Juliana Hwang; Michael H. Ing; Adler Salazar; Bernard Lassčgue; Kathy Griendling; Mohamad Navab; Alex Sevanian; Tzung K. Hsiai

2003-01-01

164

Fungal Metabolite Gliotoxin Inhibits Assembly of the Human Respiratory Burst NADPH Oxidase  

Microsoft Academic Search

Reactive oxygen species are a critical weapon in the killing of Aspergillus fumigatus by polymorphonuclear leukocytes (PMN), as demonstrated by severe aspergillosis in chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins (fumagillin, gliotoxin (GT), and helvolic acid) are examined for their effects on the NADPH oxidase activity in human PMN. Of these mycotoxins, only GT significantly and stoichiometrically

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

2004-01-01

165

The assembly of neutrophil NADPH oxidase: effects of mastoparan and its synthetic analogues.  

PubMed Central

Detergent-mediated activation of the phagocyte superoxide-generating NADPH oxidase requires the participation of at least four proteins: the membrane-bound heterodimeric cytochrome b558 and three cytosolic components, p47-phox, p67-phox and a Rac1/Rac2 protein. Peptides corresponding to sequences of different subunits of NADPH oxidase have been used as probes of the mechanism and sequence of assembly of the active complex. In the present study effects of mastoparans on activation of NADPH oxidase were investigated. Mastoparans are wasp venom cationic amphiphilic tetradecapeptides capable of modulation of various cellular activities. Natural mastoparans, as well as several synthetic mastoparan analogues, unrelated to oxidase components, blocked activation of the oxidase in the cell-free system (EC50 = 1.5 microM) and in guanosine 5'-[gamma-thio]triphosphate (GTP[S])/ATP-stimulated neutrophils permeabilized with streptolysin O. In the cell-free system the effect was not relieved by raising the detergent concentration and could not be ascribed to changes in critical micellar concentration values of the activating SDS or arachidonate. Chromatography of neutrophil cytosol on an immobilized mastoparan column suggested interaction of cytosolic p47-phox and p67-phox with the peptide. In spite of this interaction mastoparan did not interfere with translocation of p47-phox and p67-phox to the cell membranes. Images Figure 3 Figure 4

Tisch, D; Sharoni, Y; Danilenko, M; Aviram, I

1995-01-01

166

Functional analysis of the Trichoderma harzianum nox1 gene, encoding an NADPH oxidase, relates production of reactive oxygen species to specific biocontrol activity against Pythium ultimum.  

PubMed

The synthesis of reactive oxygen species (ROS) is one of the first events following pathogenic interactions in eukaryotic cells, and NADPH oxidases are involved in the formation of such ROS. The nox1 gene of Trichoderma harzianum was cloned, and its role in antagonism against phytopathogens was analyzed in nox1-overexpressed transformants. The increased levels of nox1 expression in these transformants were accompanied by an increase in ROS production during their direct confrontation with Pythium ultimum. The transformants displayed an increased hydrolytic pattern, as determined by comparing protease, cellulase, and chitinase activities with those for the wild type. In confrontation assays against P. ultimum the nox1-overexpressed transformants were more effective than the wild type, but not in assays against Botrytis cinerea or Rhizoctonia solani. A transcriptomic analysis using a Trichoderma high-density oligonucleotide (HDO) microarray also showed that, compared to gene expression for the interaction of wild-type T. harzianum and P. ultimum, genes related to protease, cellulase, and chitinase activities were differentially upregulated in the interaction of a nox1-overexpressed transformant with this pathogen. Our results show that nox1 is involved in T. harzianum ROS production and antagonism against P. ultimum. PMID:21421791

Montero-Barrientos, M; Hermosa, R; Cardoza, R E; Gutiérrez, S; Monte, E

2011-03-18

167

Regulation of NAD(P)H oxidases by AMPK in cardiovascular systems  

PubMed Central

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are ubiquitously produced in cardiovascular systems. Under physiological conditions, ROS/RNS function as signaling molecules that are essential in maintaining cardiovascular function. Aberrant concentrations of ROS/RNS have been demonstrated in cardiovascular diseases due to increased production or decreased scavenging, which have been considered as common pathways for the initiation and progression of cardiovascular diseases such as atherosclerosis, hypertension, (re)stenosis, and congestive heart failure. NAD(P)H oxidases are primary sources of ROS and can be induced or activated by all known cardiovascular risk factors. Stresses, hormones, vasoactive agents, and cytokines via different signaling cascades control the expression and activity of these enzymes and of their regulatory subunits. But the molecular mechanisms by which NAD(P)H oxidase is regulated in cardiovascular systems remain poorly characterized. Investigations by us and others suggest that adenosine monophosphate-activated protein kinase (AMPK), as an energy sensor and modulator, is highly sensitive to ROS/RNS. We have also obtained convincing evidence that AMPK is a physiological suppressor of NAD(P)H oxidase in multiple cardiovascular cell systems. In this review, we summarize our current understanding of how AMPK functions as a physiological repressor of NAD(P)H oxidase.

Song, Ping; Zou, Ming-Hui

2012-01-01

168

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

169

Inhibition of NADPH oxidase by glucosylceramide confers chemoresistance.  

PubMed

The bioactive sphingolipid ceramide induces oxidative stress by disrupting mitochondrial function and stimulating NADPH oxidase (NOX) activity, both implicated in cell death mechanisms. Many anticancer chemotherapeutics (anthracyclines, Vinca alkaloids, paclitaxel, and fenretinide), as well as physiological stimuli such as tumor necrosis factor ? (TNF?), stimulate ceramide accumulation and increase oxidative stress in malignant cells. Consequently, ceramide metabolism in malignant cells and, in particular the up-regulation of glucosylceramide synthase (GCS), has gained considerable interest in contributing to chemoresistance. We hypothesized that increases in GCS activity and thus glucosylceramide, the product of GCS activity, represents an important resistance mechanism in glioblastoma. In our study, we determined that increased GCS activity effectively blocked reactive oxygen species formation by NOX. We further showed, in both glioblastoma and neuroblastoma cells that glucosylceramide directly interfered with NOX assembly, hence delineating a direct resistance mechanism. Collectively, our findings indicated that pharmacological or molecular targeting of GCS, using non-toxic nanoliposome delivery systems, successfully augmented NOX activity, and improved the efficacy of known chemotherapeutic agents. PMID:20935456

Barth, Brian M; Gustafson, Sally J; Young, Megan M; Fox, Todd E; Shanmugavelandy, Sriram S; Kaiser, James M; Cabot, Myles C; Kester, Mark; Kuhn, Thomas B

2010-12-01

170

Inhibition of NADPH oxidase by glucosylceramide confers chemoresistance  

PubMed Central

The bioactive sphingolipid ceramide induces oxidative stress by disrupting mitochondrial function and stimulating NADPH oxidase (NOX) activity, both implicated in cell death mechanisms. Many anticancer chemotherapeutics (anthracyclines, Vinca alkaloids, paclitaxel and fenretinide), as well as physiological stimuli such as tumor necrosis factor ? (TNF?), stimulate ceramide accumulation and increase oxidative stress in malignant cells. Consequently, ceramide metabolism in malignant cells and, in particular the upregulation of glucosylceramide synthase (GCS), has gained considerable interest in contributing to chemoresistance. We hypothesized that increases in GCS activity and thus glucosylceramide, the product of GCS activity, represents an important resistance mechanism in glioblastoma. In our study, we determined that increased GCS activity effectively blocked reactive oxygen species formation by NOX. We further showed, in both glioblastoma and neuroblastoma cells that glucosylceramide directly interfered with NOX assembly, hence delineating a direct resistance mechanism. Collectively, our findings indicated that pharmacological or molecular targeting of GCS, using non-toxic nanoliposome delivery systems, successfully augmented NOX activity, and improved the efficacy of known chemotherapeutic agents.

Barth, Brian M; Gustafson, Sally J; Young, Megan M; Fox, Todd E; Shanmugavelandy, Sriram S; Kaiser, James M; Cabot, Myles C; Kuhn, Thomas B

2010-01-01

171

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.

Daugaard, Mads; Nitsch, Roberto; Razaghi, Babak; McDonald, Lindsay; Jarrar, Ameer; Torrino, Stephanie; 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

172

Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes  

PubMed Central

Reactive microglia in the CNS have been implicated in the pathogenesis of white matter disorders, such as periventricular leukomalacia and multiple sclerosis. However, the mechanism by which activated microglia kill oligodendrocytes (OLs) remains elusive. Here we show that lipopolysaccharide (LPS)-induced death of developing OLs is caused by microglia-derived peroxynitrite, the reaction product of nitric oxide (NO) and superoxide anion. Blocking peroxynitrite formation with nitric oxide synthase inhibitors, superoxide dismutase mimics, or a decomposition catalyst abrogated the cytotoxicity. Only microglia, but not OLs, expressed inducible NO synthase (iNOS) after LPS challenge; microglia from iNOS knockout mice were not cytotoxic upon activation. The molecular source for superoxide was identified as the superoxide-generating enzyme NADPH oxidase. The oxidase was activated upon LPS exposure, and its inhibition prevented microglial toxicity toward OLs. Furthermore, microglia isolated from mice deficient in the catalytic component of the oxidase, gp91phox, failed to induce cell death. Our results reveal a role for NADPH oxidase in LPS-induced OL death and suggest that peroxynitrite produced by iNOS and NADPH oxidase in activated microglia may play an important role in the pathogenesis of white matter disorders.

Li, Jianrong; Baud, Olivier; Vartanian, Timothy; Volpe, Joseph J.; Rosenberg, Paul A.

2005-01-01

173

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.

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

174

Mechanisms for suppressing NADPH oxidase in the vascular wall.  

PubMed

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 not found in leucocytes, but are highly expressed in vascular cells and upregulated with vascular remodeling, such as that found in hypertension and atherosclerosis. The difference in catalytic subunits offers the opportunity to develop "vascular specific" NADPH oxidase inhibitors that do not compromise the essential physiological signaling and phagocytic functions carried out by reactive oxygen and nitrogen species. Nitric oxide and targeted inhibitors of NADPH oxidase that block the source of oxidative stress in the vasculature are more likely to prevent the deterioration of vascular function that leads to stroke and heart attack, than are conventional antioxidants. The roles of Nox isoforms in other inflammatory conditions are yet to be explored. PMID:15962105

Dusting, Gregory J; Selemidis, Stavros; Jiang, Fan

2005-06-14

175

NADPH oxidases, reactive oxygen species, and the kidney: friend and foe.  

PubMed

Reactive oxygen species (ROS) play an important role in normal cellular physiology. They regulate different biologic processes such as cell defense, hormone synthesis and signaling, activation of G protein-coupled receptors, and ion channels and kinases/phosphatases. ROS are also important regulators of transcription factors and gene expression. On the other hand, in pathologic conditions, a surplus of ROS in tissue results in oxidative stress with various injurious consequences such as inflammation and fibrosis. NADPH oxidases are one of the many sources of ROS in biologic systems, and there are seven isoforms (Nox1-5, Duox1, Duox2). Nox4 is the predominant form in the kidney, although Nox2 is also expressed. Nox4 has been implicated in the basal production of ROS in the kidney and in pathologic conditions such as diabetic nephropathy and CKD; upregulation of Nox4 may be important in renal oxidative stress and kidney injury. Although there is growing evidence indicating the involvement of NADPH oxidase in renal pathology, there is a paucity of information on the role of NADPH oxidase in the regulation of normal renal function. Here we provide an update on the role of NADPH oxidases and ROS in renal physiology and pathology. PMID:23970124

Sedeek, Mona; Nasrallah, Rania; Touyz, Rhian M; Hébert, Richard L

2013-08-22

176

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

177

Spinal sigma-1 receptors activate NADPH oxidase 2 leading to the induction of pain hypersensitivity in mice and mechanical allodynia in neuropathic rats.  

PubMed

We have recently demonstrated that spinal sigma-1 receptors (Sig-1Rs) mediate pain hypersensitivity in mice and neuropathic pain in rats. In this study, we examine the role of NADPH oxidase 2 (Nox2)-induced reactive oxygen species (ROS) on Sig-1R-induced pain hypersensitivity and the induction of chronic neuropathic pain. Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in rats. Mechanical allodynia and thermal hyperalgesia were evaluated in mice and CCI-rats. Western blotting and dihydroethidium (DHE) staining were performed to assess the changes in Nox2 activation and ROS production in spinal cord, respectively. Direct activation of spinal Sig-1Rs with the Sig-1R agonist, PRE084 induced mechanical allodynia and thermal hyperalgesia, which were dose-dependently attenuated by pretreatment with the ROS scavenger, NAC or the Nox inhibitor, apocynin. PRE084 also induced an increase in Nox2 activation and ROS production, which were attenuated by pretreatment with the Sig-1R antagonist, BD1047 or apocynin. CCI-induced nerve injury produced an increase in Nox2 activation and ROS production in the spinal cord, all of which were attenuated by intrathecal administration with BD1047 during the induction phase of neuropathic pain. Furthermore, administration with BD1047 or apocynin reversed CCI-induced mechanical allodynia during the induction phase, but not the maintenance phase. These findings demonstrate that spinal Sig-1Rs modulate Nox2 activation and ROS production in the spinal cord, and ultimately contribute to the Sig-1R-induced pain hypersensitivity and the peripheral nerve injury-induced induction of chronic neuropathic pain. PMID:23732704

Choi, Sheu-Ran; Roh, Dae-Hyun; Yoon, Seo-Yeon; Kang, Suk-Yun; Moon, Ji-Young; Kwon, Soon-Gu; Choi, Hoon-Seong; Han, Ho-Jae; Beitz, Alvin J; Oh, Seog-Bae; Lee, Jang-Hern

2013-06-01

178

Does NADPH oxidase deficiency cause artery dilatation in humans?  

PubMed

NADPH oxidase is known to modulate the arterial tone, but the role of its specific subunits is still unclear. The objective of this study was to compare the role of p47 and gp91phox (NOX2) on artery dilatation. We conducted a multicenter study enrolling 30 patients with chronic granulomatous disease (CGD) (25 with NOX2 deficiency and 5 with p47(phox) deficiency) and 30 healthy subjects (HS), matched for gender and age, in whom flow-mediated dilation (FMD), serum activity of NOX2 (soluble NOX2-derived peptide [sNOX2-dp]), urinary isoprostanes (8-iso-PGF2?), and platelet production of isoprostanes and NOX2 were determined. Compared to HS, patients with CGD had significantly higher FMD and lower sNOX2-dp and 8-iso-PGF2? levels. Compared to patients with NOX2 deficiency and HS, patients with p47(phox) hereditary deficiency had intermediate FMD and oxidative stress, that is, higher and lower FMD and lower and higher isoprostanes compared to HS and patients with NOX2 deficiency, respectively. In agreement with this finding, an ex vivo study showed higher inhibition of NOX2 activity and lower isoprostane formation in platelets from patients with NOX2 deficiency compared to platelets from ones with p47(phox) deficiency. Our observations lead to the hypothesis that oxidants are implicated in artery vasoconstriction. PMID:23216310

Loffredo, Lorenzo; Carnevale, Roberto; Sanguigni, Valerio; Plebani, Alessandro; Rossi, Paolo; Pignata, Claudio; De Mattia, Domenico; Finocchi, Andrea; Martire, Baldassarre; Pietrogrande, Maria Cristina; Martino, Silvana; Gambineri, Eleonora; Giardino, Giuliana; Soresina, Anna Rosa; Martino, Francesco; Pignatelli, Pasquale; Violi, Francesco

2012-12-07

179

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

2012-07-27

180

Correction of aberrant NADPH oxidase activity in blood-derived mononuclear cells from type II diabetes mellitus patients by a naturally fermented papaya preparation.  

PubMed

Supplementation of standardized fermented papaya preparation (FPP) to adult diabetic mice improves dermal wound healing outcomes. Peripheral blood mononuclear cells (PBMC) from type II diabetes mellitus (T2DM) patients elicit a compromised respiratory burst activity resulting in increased risk of infections for the diabetic patients. Aims: The objectives of the current study were to determine the effect of FPP supplementation on human diabetic PBMC respiratory burst activity and to understand underlying mechanisms of such action of FPP. Results: When stimulated with phorbol 12-myristate 13-acetate, the production of reactive oxygen species by T2DM PBMC was markedly compromised compared to that of the PBMC from non-DM donors. FPP treated ex vivo improved respiratory burst outcomes in T2DM PBMC. FPP treatment significantly increased phosphorylation of the p47phox subunit of NADPH oxidase. In addition, the protein and mRNA expression of Rac2 was potently upregulated after FPP supplemention. The proximal human Rac2 gene promoter is G-C rich and contains consensus binding sites for Sp1 and AP-1. While FPP had no significant effect on the AP-1 DNA binding activity, the Sp1 DNA binding activity was significantly upregulated in PBMC after treatment of the cells with FPP. Innovation: This work provided first evidence that compromised respiratory burst performance of T2DM PBMC may be corrected by a nutritional supplement. Conclusion: FPP can correct respiratory burst performance of T2DM PBMC via an Sp-1-dependant pathway. Studies testing the outcome of FPP supplementation in diabetic patients are warranted. PMID:22369197

Dickerson, Ryan; Deshpande, Bhakthi; Gnyawali, Urmila; Lynch, Debbie; Gordillo, Gayle M; Schuster, Dara; Osei, Kwame; Roy, Sashwati

2012-04-26

181

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

2011-11-18

182

Homocysteine induces VCAM-1 gene expression through NF-kappaB and NAD(P)H oxidase activation: protective role of Mediterranean diet polyphenolic antioxidants.  

PubMed

Hyperhomocysteinemia is a recognized risk factor for vascular disease, but pathogenetic mechanisms involved in its vascular actions are largely unknown. Because VCAM-1 expression is crucial in monocyte adhesion and early atherogenesis, we evaluated the NF-kappaB-related induction of VCAM-1 by homocysteine (Hcy) and the possible inhibitory effect of dietary polyphenolic antioxidants, such as trans-resveratrol (RSV) and hydroxytyrosol (HT), which are known inhibitors of NF-kappaB-mediated VCAM-1 induction. In human umbilical vein endothelial cells (HUVEC), Hcy, at 100 micromol/l, but not cysteine, induced VCAM-1 expression at the protein and mRNA levels, as shown by enzyme immunoassay and Northern analysis, respectively. Transfection studies with deletional VCAM-1 promoter constructs demonstrated that the two tandem NF-kappaB motifs in the VCAM-1 promoter are necessary for Hcy-induced VCAM-1 gene expression. Hcy-induced NF-kappaB activation was confirmed by EMSA, as shown by the nuclear translocation of its p65 (RelA) subunit and the degradation of the inhibitors IkappaB-alpha and IkappaB-beta by Western analysis. Hcy also increased intracellular reactive oxygen species by NAD(P)H oxidase activation, as shown by the membrane translocation of its p47(phox) subunit. NF-kappaB inhibitors decreased Hcy-induced intracellular reactive oxygen species and VCAM-1 expression. Finally, we found that nutritionally relevant concentrations of RSV and HT, but not folate and vitamin B6, reduce (by >60% at 10(-6) mol/l) Hcy-induced VCAM-1 expression and monocytoid cell adhesion to the endothelium. These data indicate that pathophysiologically relevant Hcy concentrations induce VCAM-1 expression through a prooxidant mechanism involving NF-kappaB. Natural Mediterranean diet antioxidants can inhibit such activation, suggesting their possible therapeutic role in Hcy-induced vascular damage. PMID:17586618

Carluccio, Maria Annunziata; Ancora, Maria Assunta; Massaro, Marika; Carluccio, Marisa; Scoditti, Egeria; Distante, Alessandro; Storelli, Carlo; De Caterina, Raffaele

2007-06-22

183

Role of the NADPH oxidases in the subfornical organ in angiotensin II-induced hypertension.  

PubMed

Reactive oxygen species and the NADPH oxidases contribute to hypertension via mechanisms that remain undefined. Reactive oxygen species produced in the central nervous system have been proposed to promote sympathetic outflow, inflammation, and hypertension, but the contribution of the NADPH oxidases to these processes in chronic hypertension is uncertain. We therefore sought to identify how NADPH oxidases in the subfornical organ (SFO) of the brain regulate blood pressure and vascular inflammation during sustained hypertension. We produced mice with loxP sites flanking the coding region of the NADPH oxidase docking subunit p22(phox). SFO-targeted injections of an adenovirus encoding cre-recombinase markedly diminished p22(phox), Nox2, and Nox4 mRNA in the SFO, as compared with a control adenovirus encoding red-fluorescent protein injection. Increased superoxide production in the SFO by chronic angiotensin II infusion (490 ng/kg min(-1) × 2 weeks) was blunted in adenovirus encoding cre-recombinase-treated mice, as detected by dihydroethidium fluorescence. Deletion of p22(phox) in the SFO eliminated the hypertensive response observed at 2 weeks of angiotensin II infusion compared with control adenovirus encoding red-fluorescent protein-treated mice (mean arterial pressures=97 ± 15 versus 154 ± 6 mm Hg, respectively; P=0.0001). Angiotensin II infusion also promoted marked vascular inflammation, as characterized by accumulation of activated T-cells and other leukocytes, and this was prevented by deletion of the SFO p22(phox). These experiments definitively identify the NADPH oxidases in the SFO as a critical determinant of the blood pressure and vascular inflammatory responses to chronic angiotensin II, and further support a role of reactive oxygen species in central nervous system signaling in hypertension. PMID:23248154

Lob, Heinrich E; Schultz, David; Marvar, Paul J; Davisson, Robin L; Harrison, David G

2012-12-17

184

The NADPH oxidase of professional phagocytes--prototype of the NOX electron transport chain systems  

PubMed Central

The NADPH oxidase is an electron transport chain in “professional” phagocytic cells that transfers electrons from NADPH in the cytoplasm, across the wall of the phagocytic vacuole, to form superoxide. The electron transporting flavocytochrome b is activated by the integrated function of four cytoplasmic proteins. The antimicrobial function of this system involves pumping K+ into the vacuole through BKCa channels, the effect of which is to elevate the vacuolar pH and activate neutral proteases. A number of homologous systems have been discovered in plants and lower animals as well as in man. Their function remains to be established.

Cross, Andrew R.; Segal, Anthony W.

2009-01-01

185

The NADPH oxidase inhibitor apocynin (acetovanillone) induces oxidative stress  

SciTech Connect

Apocynin (acetovanillone) is often used as a specific inhibitor of NADPH oxidase. In N11 glial cells, apocynin induced, in a dose-dependent way, a significant increase of both malonyldialdehyde level (index of lipid peroxidation) and lactate dehydrogenase release (index of a cytotoxic effect). Apocynin evoked also, in a significant way, an increase of H{sub 2}O{sub 2} concentration and a decrease of the intracellular glutathione/glutathione disulfide ratio, accompanied by augmented efflux of glutathione and glutathione disulfide. Apocynin induced the activation of both pentose phosphate pathway and tricarboxylic acid cycle, which was blocked when the cells were incubated with glutathione together with apocynin. The cell incubation with glutathione prevented also the apocynin-induced increase of malonyldialdehyde generation and lactate dehydrogenase leakage. Apocynin exerted an oxidant effect also in a cell-free system: indeed, in aqueous solution, it evoked a faster oxidation of the thiols glutathione and dithiothreitol, and elicited the generation of reactive oxygen species, mainly superoxide anions. Our results suggest that apocynin per se can induce an oxidative stress and exert a cytotoxic effect in N11 cells and other cell types, and that some effects of apocynin in in vitro and in vivo experimental models should be interpreted with caution.

Riganti, Chiara [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Turin (Italy); Costamagna, Costanzo [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Turin (Italy); Bosia, Amalia [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Turin (Italy); Ghigo, Dario [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Turin (Italy)]. E-mail: dario.ghigo@unito.it

2006-05-01

186

Molecular cloning and sequencing of Japanese pufferfish ( Takifugu rubripes) NADPH oxidase cDNAs  

Microsoft Academic Search

The superoxide-producing NADPH oxidase complex of phagocytes plays a crucial role in host defenses against microbial infection. NADPH oxidase consists of a membrane heterodimeric protein, composed of gp91phox and p22phox, and cytosolic proteins, p40phox, p47phox and p67phox.In the present study, cDNAs of all the components of NADPH oxidase were cloned from peripheral white blood cells of the Japanese pufferfish utilizing

Yuuki Inoue; Yuuki Suenaga; Yasutoshi Yoshiura; Tadaaki Moritomo; Mitsuru Ototake; Teruyuki Nakanishi

2004-01-01

187

Opening the black box: Lessons from cell-free systems on the phagocyte NADPH-oxidase  

Microsoft Academic Search

The NADPH-oxidase complex of phagocytic cells plays a key role in the defense against invading pathogens, through the release of superoxide anion, precursor of other reactive oxygen species (ROS). NADPH-oxidase deficiency is called Chronic Granulomatous Disease (CGD), in which patients suffer from recurrent infections and from the formation of granulomas in various organs. Research on NADPH-oxidase has much benefited from

Marie-Claire Dagher; Edgar Pick

2007-01-01

188

Biphasic regulation of the NADPH oxidase by HGF/c-Met signaling pathway in primary mouse hepatocytes.  

PubMed

Redox signaling is emerging as an essential mechanism in the regulation of biological activities of the cell. The HGF/c-Met signaling pathway has been implicated as a key regulator of the cellular redox homeostasis and oxidative stress. We previously demonstrated that genetic deletion of c-Met in hepatocytes disrupts redox homeostasis by a mechanism involving NADPH oxidase. Here, we were focused to address the mechanism of NADPH oxidase regulation by HGF/c-Met signaling in primary mouse hepatocytes and its relevance. HGF induced a biphasic mechanism of NADPH oxidase regulation. The first phase employed the rapid increase in production of ROS as signaling effectors to activate the Nrf2-mediated protective response resulting in up-regulation of the antioxidant proteins, such as NAD(P)H quinone oxidoreductase and ?-glutamylcysteine synthetase. The second phase operated under a prolonged HGF exposure, caused a suppression of the NADPH oxidase components, including NOX2, NOX4, p22 and p67, and was able to abrogate the TGF?-induced ROS production and improve cell viability. In conclusion, HGF/c-Met induces a Nrf2-mediated protective response by a double mechanism driven by NADPH oxidase. PMID:23333744

Clavijo-Cornejo, Denise; Enriquez-Cortina, Cristina; López-Reyes, Alberto; Domínguez-Pérez, Mayra; Nuńo, Natalia; Domínguez-Meraz, Marcela; Bucio, Leticia; Souza, Verónica; Factor, Valentina M; Thorgeirsson, Snorri S; Gutiérrez-Ruiz, María Concepción; Gómez-Quiroz, Luis E

2013-01-16

189

Selective Rac1 inhibition protects renal tubular epithelial cells from oxalate-induced NADPH oxidase-mediated oxidative cell injury  

PubMed Central

Oxalate-induced oxidative cell injury is one of the major mechanisms implicated in calcium oxalate nucleation, aggregation and growth of kidney stones. We previously demonstrated that oxalate-induced NADPH oxidase-derived free radicals play a significant role in renal injury. Since NADPH oxidase activation requires several regulatory proteins, the primary goal of this study was to characterize the role of Rac GTPase in oxalate-induced NADPH oxidase-mediated oxidative injury in renal epithelial cells. Our results show that oxalate significantly increased membrane translocation of Rac1 and NADPH oxidase activity of renal epithelial cells in a time-dependent manner. We found that NSC23766, a selective inhibitor of Rac1, blocked oxalate-induced membrane translocation of Rac1 and NADPH oxidase activity. In the absence of Rac1 inhibitor, oxalate exposure significantly increased hydrogen peroxide formation and LDH release in renal epithelial cells. In contrast, Rac1 inhibitor pretreatment, significantly decreased oxalate-induced hydrogen peroxide production and LDH release. Furthermore, PKC ? and ? inhibitor, oxalate exposure did not increase Rac1 protein translocation, suggesting that PKC resides upstream from Rac1 in the pathway that regulates NADPH oxidase. In conclusion, our data demonstrate for the first time that Rac1-dependent activation of NADPH oxidase might be a crucial mechanism responsible for oxalate-induced oxidative renal cell injury. These findings suggest that Rac1 signaling plays a key role in oxalate-induced renal injury, and may serve as a potential therapeutic target to prevent calcium oxalate crystal deposition in stone formers and reduce recurrence.

Thamilselvan, Vijayalakshmi; Menon, Mani

2013-01-01

190

ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation  

PubMed Central

Pollen exposure induces allergic airway inflammation in sensitized subjects. The role of antigenic pollen proteins in the induction of allergic airway inflammation is well characterized, but the contribution of other constituents in pollen grains to this process is unknown. Here we show that pollen grains and their extracts contain intrinsic NADPH oxidases. The pollen NADPH oxidases rapidly increased the levels of ROS in lung epithelium as well as the amount of oxidized glutathione (GSSG) and 4-hydroxynonenal (4-HNE) in airway-lining fluid. These oxidases, as well as products of oxidative stress (such as GSSG and 4-HNE) generated by these enzymes, induced neutrophil recruitment to the airways independent of the adaptive immune response. Removal of pollen NADPH oxidase activity from the challenge material reduced antigen-induced allergic airway inflammation, the number of mucin-containing cells in airway epithelium, and antigen-specific IgE levels in sensitized mice. Furthermore, challenge with Amb a 1, the major antigen in ragweed pollen extract that does not possess NADPH oxidase activity, induced low-grade allergic airway inflammation. Addition of GSSG or 4-HNE to Amb a 1 challenge material boosted allergic airway inflammation. We propose that oxidative stress generated by pollen NADPH oxidases (signal 1) augments allergic airway inflammation induced by pollen antigen (signal 2).

Boldogh, Istvan; Bacsi, Attila; Choudhury, Barun K.; Dharajiya, Nilesh; Alam, Rafeul; Hazra, Tapas K.; Mitra, Sankar; Goldblum, Randall M.; Sur, Sanjiv

2005-01-01

191

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

PubMed

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; Touyz, Rhian M

2012-06-11

192

5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts.  

PubMed Central

The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5-HT)(1A) receptor, which activates the extracellular signal-regulated kinase (ERK) through a G(i)betagamma-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT(1A) receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT(1A) receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT(1A) receptor in Chinese hamster ovary (CHO) cells downstream of G(i)betagamma subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT(1A) receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide.

Mukhin, Y V; Garnovskaya, M N; Collinsworth, G; Grewal, J S; Pendergrass, D; Nagai, T; Pinckney, S; Greene, E L; Raymond, J R

2000-01-01

193

NADPH oxidase-derived free radicals are key oxidants in alcohol-induced liver disease  

PubMed Central

In North America, liver disease due to alcohol consumption is an important cause of death in adults, although its pathogenesis remains obscure. Despite the fact that resident hepatic macrophages are known to contribute to early alcohol-induced liver injury via oxidative stress, the exact source of free radicals has remained a mystery. To test the hypothesis that NADPH oxidase is the major source of oxidants due to ethanol, we used p47phox knockout mice, which lack a critical subunit of this major source of reactive oxygen species in activated phagocytes. Mice were treated with ethanol chronically, using a Tsukamoto-French protocol, for 4 weeks. In wild-type mice, ethanol caused severe liver injury via a mechanism involving gut-derived endotoxin, CD14 receptor, production of electron spin resonance–detectable free radicals, activation of the transcription factor NF-?B, and release of cytotoxic TNF-? from activated Kupffer cells. In NADPH oxidase–deficient mice, neither an increase in free radical production, activation of NF-?B, an increase in TNF-? mRNA, nor liver pathology was observed. These data strongly support the hypothesis that free radicals from NADPH oxidase in hepatic Kupffer cells play a predominant role in the pathogenesis of early alcohol-induced hepatitis by activating NF-?B, which activates production of cytotoxic TNF-?.

Kono, Hiroshi; Rusyn, Ivan; Yin, Ming; Gabele, Erwin; Yamashina, Shunhei; Dikalova, Anna; Kadiiska, Maria B.; Connor, Henry D.; Mason, Ronald P.; Segal, Brahm H.; Bradford, Blair U.; Holland, Steven M.; Thurman, Ronald G.

2000-01-01

194

NADPH oxidase inhibits the pathogenesis of systemic lupus erythematosus.  

PubMed

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by loss of tolerance to self nucleic acids. The source of autoantigen that drives disease onset and progression is unclear. A candidate source of autoantigen is the neutrophil extracellular trap (NET), which releases nucleic acids into the extracellular environment, generating a structure composed of DNA coated with antimicrobial proteins. On the basis of in vitro and patient correlative studies, several groups have suggested that NETs may provide lupus autoantigens. The observation that NET release (NETosis) relies on activity of the phagocyte NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (Nox2) in neutrophils of both humans and mice provided a genetic strategy to test this hypothesis in vivo. Therefore, we crossed an X-linked nox2 null allele onto the lupus-prone MRL.Fas(lpr) genetic background and assessed immune activation, autoantibody generation, and SLE pathology. Counter to the prevailing hypothesis, Nox2-deficient lupus-prone mice had markedly exacerbated lupus, including increased spleen weight, increased renal disease, and elevated and altered autoantibody profiles. Moreover, heterozygous female mice, which have Nox2 deficiency in 50% of neutrophils, also had exacerbated lupus and altered autoantibody patterns, suggesting that failure to undergo normal Nox2-dependent cell death may result in release of immunogenic self-constituents that stimulate lupus. Our results indicate that NETosis does not contribute to SLE in vivo; instead, Nox2 acts to inhibit disease pathogenesis, making this enzyme an important target for further study and a candidate for therapeutic intervention. PMID:23100627

Campbell, Allison M; Kashgarian, Michael; Shlomchik, Mark J

2012-10-24

195

NADPH oxidase-derived reactive oxygen species in the regulation of endothelial phenotype.  

PubMed

Endothelial dysfunction comprising impairment of endothelium-dependent vasodilator function and increased endothelial activation contributes to the pathophysiology of cardiovascular diseases such as atherosclerosis, diabetic vasculopathy, heart failure and hypertension. The changes in endothelial phenotype in these conditions occur in response to diverse stimuli including inflammatory cytokines, activation of renin-angiotensin-aldosterone system, hyperlipidaemia, hyperglycemia, ischemia-reperfusion and mechanical forces. An increased production of reactive oxygen species (ROS), such as superoxide and H(2)O(2) is involved in the genesis of these alterations in endothelial phenotype. The NADPH oxidases, Nox2 and Nox4, are major sources of ROS in endothelial cells and are implicated both in vasodilator dysfunction and in the modulation of redox-sensitive signalling pathways that influence endothelial cytoskeletal organisation, adhesion molecule expression, permeability, growth, migration and other functions. NADPH oxidases appear to be especially important in redox signalling in that they are specifically activated by diverse agonists and regulate the activation of downstream protein kinases, transcription factors and other biological molecules. This review provides an overview of NADPH oxidase structure and regulation in endothelial cells and their role in pathophysiology, focussing particularly on endothelial activation. PMID:18276982

Dworakowski, Rafa?; Alom-Ruiz, Sara P; Shah, Ajay M

196

Voluntary wheel running restores endothelial function in conduit arteries of old mice: direct evidence for reduced oxidative stress, increased superoxide dismutase activity and down-regulation of NADPH oxidase  

PubMed Central

Habitual aerobic exercise is associated with enhanced endothelium-dependent dilatation (EDD) in older humans, possibly by increasing nitric oxide bioavailability and reducing oxidative stress. However, the mechanisms involved are incompletely understood. EDD was measured in young (6–8 months) and old (29–32 months) cage control and voluntary wheel running (VR) B6D2F1 mice. Age-related reductions in maximal carotid artery EDD to acetylcholine (74 vs. 96%, P < 0.01) and the nitric oxide (NO) component of EDD (maximum dilatation with ACh and l-NAME minus that with ACh alone was ?28%vs.?55%, P < 0.01) were restored in old VR (EDD: 96%, NO: ?46%). Nitrotyrosine, a marker of oxidative stress, was increased in aorta with age, but was markedly lower in old VR (P < 0.05). Aortic superoxide dismutase (SOD) activity was greater (P < 0.01), whereas NADPH oxidase protein expression (P < 0.01) and activity (P= 0.05) were lower in old VR vs. old cage control. Increasing SOD (with 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl) and inhibition of NADPH oxidase (with apocynin) improved EDD and its NO component in old cage control, but not old VR mice. VR increased endothelial NO synthase (eNOS) protein expression (P < 0.05) and activation (Ser1177 phosphorylation) (P < 0.05) in old mice. VR did not affect EDD in young mice. Our results show that voluntary aerobic exercise restores the age-associated loss of EDD by suppression of oxidative stress via stimulation of SOD antioxidant activity and inhibition of NADPH oxidase superoxide production. Increased eNOS protein and activation also may contribute to exercise-mediated preservation of NO bioavailability and EDD with ageing.

Durrant, Jessica R; Seals, Douglas R; Connell, Melanie L; Russell, Molly J; Lawson, Brooke R; Folian, Brian J; Donato, Anthony J; Lesniewski, Lisa A

2009-01-01

197

Squamosamide derivative FLZ protects dopaminergic neurons against inflammation-mediated neurodegeneration through the inhibition of NADPH oxidase activity  

Microsoft Academic Search

BACKGROUND: Inflammation plays an important role in the pathogenesis of Parkinson's disease (PD) through over-activation of microglia, which consequently causes the excessive production of proinflammatory and neurotoxic factors, and impacts surrounding neurons and eventually induces neurodegeneration. Hence, prevention of microglial over-activation has been shown to be a prime target for the development of therapeutic agents for inflammation-mediated neurodegenerative diseases. METHODS:

Dan Zhang; Xiaoming Hu; Sung-Jen Wei; Jie Liu; Huiming Gao; Li Qian; Belinda Wilson; Gengtao Liu; Jau-Shyong Hong

2008-01-01

198

Arachidonic Acid Activates c-Jun N-Terminal Kinase through NADPH Oxidase in Rabbit Proximal Tubular Epithelial Cells  

Microsoft Academic Search

In kidney epithelial cells, arachidonic acid and other fatty acids are important signal transduction molecules for G protein-coupled receptors. We now demonstrate that arachidonic acid induced a time- and dose-dependent activation of JNK, a member of the mitogenactivated protein kinase family, as assessed by phosphorylation of the transcription factor ATF-2. Increments in JNK activity were detectable at 5 mu M

Xiao-Lan Cui; Janice G. Douglas

1997-01-01

199

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

Microsoft Academic Search

(LV) 1 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 distalpulmonaryvasculature. CIH-inducedpulmonary hypertension was associated with increased lung levels of the NADPH oxidase subunits, Nox4 and p22phox, as well as increased activity of

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

2008-01-01

200

Pulsatile Versus Oscillatory Shear Stress Regulates NADPH Oxidase Subunit Expression Implication for Native LDL Oxidation  

Microsoft Academic Search

Shear stress regulates endothelial nitric oxide and superoxide (O 2·) production, implicating the role of NADPH oxidase activity. It is unknown whether shear stress regulates the sources of reactive species production, consequent low-density lipoprotein (LDL) modification, and initiation of inflammatory events. Bovine aortic endothelial cells (BAECs) in the presence of 50 g\\/mL of native LDL were exposed to (1) pulsatile

Juliana Hwang; Michael H. Ing; Adler Salazar; Bernard Lassčgue; Kathy Griendling; Mohamad Navab; Alex Sevanian; Tzung K. Hsiai

2010-01-01

201

Hypoxia-Dependent Regulation of Nonphagocytic NADPH Oxidase Subunit NOX4 in the Pulmonary Vasculature  

Microsoft Academic Search

Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Chronic hypoxia induces vascular remodeling with medial hypertrophy leading to the development of pulmonary hypertension. We

Manish Mittal; Markus Roth; Peter Konig; Simone Hofmann; Eva Dony; Parag Goyal; Anne-Christin Selbitz; Ralph Theo Schermuly; Grazyna Kwapiszewska; Wolfgang Kummer; Walter Klepetko; Mir Ali Reza Hoda; Ludger Fink; Jorg Hanze; Werner Seeger; Friedrich Grimminger; Harald H. H. W. Schmidt; Norbert Weissmann

2010-01-01

202

Identification and Characterization of Sclerotinia sclerotiorum NADPH Oxidases?†  

PubMed Central

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. ?Ssnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum.

Kim, Hyo-jin; Chen, Changbin; Kabbage, Mehdi; Dickman, Martin B.

2011-01-01

203

Post-treatment of an NADPH oxidase inhibitor prevents seizure-induced neuronal death.  

PubMed

The present study sought to evaluate the neuroprotective effects of apocynin, an NADPH oxidase assembly inhibitor, on seizure-induced neuronal death. Apocynin, also known as acetovanillone, is a natural organic compound isolated from the root of Canadian hemp (Apocynum cannabium). It has been extensively studied to determine its disease-fighting capabilities and application in several brain insults, such as traumatic brain injury and stroke. Here we tested the hypothesis that post-treatment of apocynin may prevent seizure-induced neuronal death by suppression of NADPH oxidase-mediated superoxide production. Temporal lobe epilepsy (TLE) was induced by intraperitoneal injection of pilocarpine (25mg/kg) in male rats. Apocynin (30mg/kg, i.p.) was injected into the intraperitoneal space two hours after seizure onset. A second injection was performed 24h after seizure. To test whether apocynin inhibits NADPH oxidase activation-induced reactive oxygen species (ROS) production, dihydroethidium (dHEt, 5mg/kg, i.p.) was injected before onset of seizure and ROS production was detected five hours after seizure onset. Neuronal oxidative injury (4HNE), neuronal death (Fluoro Jade-B), blood brain barrier (BBB) disruption (IgG leak), neurotrophil infiltration (MPO) and microglia activation (CD11b) in the hippocampus was evaluated at three days after status epilepticus (SE). Pilocarpine-induced seizure increased p47 immunofluorescence in the plasma membrane of hippocampal neurons at 12h post-insult and apocynin treatment prevented this increase. The present study found that apocynin post-treatment decreased ROS production and lipid peroxidation after seizure and decreased the number of degenerating hippocampal neurons. Apocynin also reduced seizure-induced BBB disruption, neurotrophil infiltration and microglial activation. Taken together, the present results suggest that inhibition of NADPH oxidase by apocynin may have a high therapeutic potential to reduce seizure-induced neuronal dysfunction. PMID:23313582

Kim, Jin Hee; Jang, Bong Geom; Choi, Bo Young; Kim, Hyeong Seop; Sohn, Min; Chung, Tae Nyoung; Choi, Hui Chul; Song, Hong Ki; Suh, Sang Won

2013-01-10

204

Role of apoptosis-inducing factor, proline dehydrogenase, and NADPH oxidase in apoptosis and oxidative stress  

PubMed Central

Flavoproteins catalyze a variety of reactions utilizing flavin mononucleotide or flavin adenine dinucleotide as cofactors. The oxidoreductase properties of flavoenzymes implicate them in redox homeostasis, oxidative stress, and various cellular processes, including programmed cell death. Here we explore three critical flavoproteins involved in apoptosis and redox signaling, ie, apoptosis-inducing factor (AIF), proline dehydrogenase, and NADPH oxidase. These proteins have diverse biochemical functions and influence apoptotic signaling by unique mechanisms. The role of AIF in apoptotic signaling is two-fold, with AIF changing intracellular location from the inner mitochondrial membrane space to the nucleus upon exposure of cells to apoptotic stimuli. In the mitochondria, AIF enhances mitochondrial bioenergetics and complex I activity/assembly to help maintain proper cellular redox homeostasis. After translocating to the nucleus, AIF forms a chromatin degrading complex with other proteins, such as cyclophilin A. AIF translocation from the mitochondria to the nucleus is triggered by oxidative stress, implicating AIF as a mitochondrial redox sensor. Proline dehydrogenase is a membrane-associated flavoenzyme in the mitochondrion that catalyzes the rate-limiting step of proline oxidation. Upregulation of proline dehydrogenase by the tumor suppressor, p53, leads to enhanced mitochondrial reactive oxygen species that induce the intrinsic apoptotic pathway. NADPH oxidases are a group of enzymes that generate reactive oxygen species for oxidative stress and signaling purposes. Upon activation, NADPH oxidase 2 generates a burst of superoxide in neutrophils that leads to killing of microbes during phagocytosis. NADPH oxidases also participate in redox signaling that involves hydrogen peroxide-mediated activation of different pathways regulating cell proliferation and cell death. Potential therapeutic strategies for each enzyme are also highlighted.

Natarajan, Sathish Kumar; Becker, Donald F

2012-01-01

205

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.

van der Vliet, Albert

2008-01-01

206

Effects of anti-allergic drugs on human neutrophil superoxide-generating NADPH oxidase.  

PubMed

The effects of anti-allergic drugs with or without H1-receptor antagonism on the NADPH oxidase (EC 1.6.99.6) from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Three anti-allergic drugs with H1-receptor antagonism, azelastine, ketotifen and oxatomide, were found to inhibit the superoxide generation of human neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of these drugs required for 50% inhibition of the oxidase (IC50) were: azelastine--0.7 microM in the whole-cell system and 0.5 microM in the cell-free system; ketotifen--60 microM in the whole-cell system and 6.8 microM in the cell-free system; and oxatomide--25 microM in the whole-cell system and 9.7 microM in the cell-free system. In addition, in the cell-free system, these drugs did not change the Km values for the NADPH of the oxidase. However, these drugs did not inhibit the superoxide generation of NADPH oxidase after its activation in whole-cell and cell-free systems, suggesting that these drugs do not have superoxide-scavenger actions. Concentrations of less than 200 microM of anti-allergic drugs without H1-receptor antagonism, tranilast, repirinast and ibudilast, did not inhibit neutrophil NADPH oxidase in whole-cell and cell-free systems. The IC50 of hydrocortisone in the cell-free system was 60 microM. These results suggest that anti-allergic drugs with H1-receptor antagonism inhibit activation of the solubilized membrane-bound enzyme by sodium dodecyl sulfate in cell-free systems and that they have much stronger anti-inflammatory action than hydrocortisone. PMID:1372806

Umeki, S

1992-03-01

207

The role of NADPH oxidase in chronic intermittent hypoxia-induced pulmonary hypertension in mice.  

PubMed

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 beta 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

2008-10-23

208

Regulation of NADPH oxidase gene expression with PKA and cytokine IL-4 in neurons and microglia.  

PubMed

Neuronal excitation is mediated by the activation of NMDA receptor and associated with the formation of reactive oxygen species due to the activation of NADPH oxidase complex proteins. The activation of Gs protein coupled receptors (GPCRs) induces neuronal activation in the cAMP-dependent protein kinase A (PKA)-mediated signal cascade and regulates NADPH oxidase activity. However, it is unknown whether PKA regulates NADPH oxidase gene expression in neurons and microglia. In the present research, the NADPH oxidase gene expression was studied in rat cortical neurons and microglia in vitro. Purified microglial cells were identified with OX-42 antibody and they also expressed apolipoprotein E (ApoE). The time-dependent effect of cytokine interleukin-4 (IL-4) (20 ng/ml) in NADPH oxidase gene expression was studied in microglial cells. The levels of mRNA were determined by quantitative RT-PCR. The expression of NOX1, NOX2, and NCF2 was upregulated after IL-4 treatment for 4 h, but it was downregulated after 8-24 h. The expression of NCF1 was suppressed during any time of cytokine effect. IL-4 upregulated arginase1 (Arg1) and serine racemase1 (SRR1) gene expressions in microglia. Amyloid beta (Ab) suppressed NOX2, NCF1, and NCF2 gene expressions and upregulated glutamate cystine transporter (xCT), although IL-4 attenuated the effect of Ab (500 ?M) in the upregulation of xCT gene expression. The activation of PKA with agonist dibutyryl cAMP (dbcAMP) (100 ?M) induced the upregulation of Arg1 gene expression in microglia involving in the process of microglial activation. The transcription of NOX1, NOX2, and NCF1 was suppressed in microglial cells after dbcAMP treatment within 24 h. Neurons were identified with the microtubule-associated protein tau. The uniform distribution of tau along axons was established in normal neurons. Tau protein was redistributed after PKA agonist dbcAMP treatment for 24 h. L-glutamate (50 ?M) caused the apoptotic processes and the accumulation of tau in the soma of neurons and along axons. The activation of PKA for 24 h induced the transcriptional upregulation of NOX1 and NCF1 in cortical neurons. However, L-glutamate suppressed NOX1 gene expression in neurons. These data demonstrate that the effects of IL-4 and dbcAMP are similar in the regulation of SRR1, Arg1, and NADPH oxidase complex gene expressions in neurons and microglia. IL-4 prevents glutamate release from microglia suppressing xCT expression induced by Ab. These findings suggest that the activation of GPCR in PKA-mediated pathway leads to transcriptional regulation of NADPH oxidase complex. The modulation of GPCR activation may inhibit the oxidative stress in neurons. PMID:22565378

Savchenko, Valentina L

2012-05-08

209

NADPH oxidase isoforms and anti-hypertensive effects of atorvastatin demonstrated in two animal models.  

PubMed

Beneficial effects of statins on cardiovascular diseases have been attributed to decreased generation of reactive oxygen species (ROS). We tested the hypothesis that atorvastatin protects against the development of hypertension by reducing levels of NADPH oxidase-derived ROS in two hypertensive animal models. Atorvastatin was given to mice chronically infused with angiotensin (Ang) II or to apolipoprotein E (ApoE)-deficient mice fed a high-fat diet. Increased mean blood pressure (MBP) demonstrated in both animal models was significantly suppressed by atorvastatin with reduced ROS production in the aorta. Treatment with atorvastatin did not alter the mRNA level of NOX1, a catalytic subunit of NADPH oxidase, but decreased the levels of other NOX isoforms, NOX2 and NOX4, in the ApoE-deficient mice fed a high-fat diet. In the Ang II-infused model treated with statin, only the NOX4 mRNA level was reduced. Membrane translocation of Rac1 was significantly reduced in the Ang II-infused mice treated with atorvastatin. Finally, atorvastatin administered to Ang II-infused mice lacking the Nox1 gene elicited an additional decline in MBP compared to Nox1-deficient mice treated with vehicle. Together, these findings suggest that reduced expression and activity of the isoforms of NADPH oxidase, involving NOX1, NOX2, and possibly NOX4, mediate the anti-hypertensive effect of atorvastatin. PMID:19881226

Cui, Wenhao; Matsuno, Kuniharu; Iwata, Kazumi; Ibi, Masakazu; Katsuyama, Masato; Kakehi, Tomoko; Sasaki, Mika; Ikami, Kanako; Zhu, Kai; Yabe-Nishimura, Chihiro

2009-10-31

210

Oxytocin inhibits NADPH oxidase and P38 MAPK in cisplatin-induced nephrotoxicity.  

PubMed

Oxidative stress significantly contributes to cisplatin (CP)-associated cytotoxicity, and use of antioxidants could counteract such cytotoxic effects of CP. The major biochemical pathway for reactive oxygen species (ROS) formation proceeds through O?? production, which is generated by NADPH oxidase, such oxidative stress can activate p38 MAPK to intensify the cytotoxic effect of CP. We mainly aimed to study the protective effect of oxytocin (OT) on CP-induced nephrotoxicity whereas; it was previously shown to have anti-inflammatory effects in different inflammation models. Administration of OT significantly decreased the gene expression of both NADPH oxidase and P38 MAPK, nitric oxide (NO), myloperoxidase (MPO), and TBARS, furthermore it increased the renal tissue levels of antioxidants; reduced glutathione (GSH), and superoxide dismutase (SOD). Histologically, OT reduced the monocellular infiltration as well as the tubular damage in CP-induced nephrotoxicity. In conclusion OT has a powerful antioxidant effect that can alleviate the CP-induced nephrotoxicity through inhibition of NADPH oxidase and P38 MAPK resulting in improvement of kidney functions. PMID:21993003

Rashed, Laila A; Hashem, Reem M; Soliman, Hala M

2011-08-27

211

Potential role of the "NADPH oxidases" (NOX/DUOX) family in cystic fibrosis.  

PubMed

Cystic fibrosis (CF), is the most common life-shortening autosomal recessive disorder in Caucasians. It is caused by mutations in a single gene on the long arm of chromosome 7 that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CF is characterized by abnormal Na+ and Cl- ion transport in several tissues, including the lungs, pancreas, gastrointestinal tract, liver, sweat glands, and male reproductive system. Progressive pulmonary disease is the dominant clinical feature of CF and accounts for morbidity and mortality. The inflammation characterized by an overabundance of activated neutrophils and macrophages on the respiratory epithelial surface is associated to a high production of reactive oxygen species (ROS) which contribute to the pathogenesis of cystic fibrosis. ROS could have different origins but the role of the NADPH oxidase system is essential. The "NADPH oxidases" (NOX/DUOX) family is an enzymatic complex formed by cytosolic and membrane subunits. Until now several homologues of the phagocytic NADPH oxidase have been identified in different tissues and it has been shown that the lungs preferentially expressed DUOX1-2. Thus, DUOX1-2 could be implicated in the anti-infectious defense system. The role of DUOX enzymes as a source of ROS in cystic fibrosis is examined as they could contribute to a better understanding of molecular mechanisms in CF. Moreover they could be a potential target for a new therapeutic approach. PMID:19091660

Pongnimitprasert, N; El-Benna, J; Foglietti, M J; Gougerot-Pocidalo, M A; Bernard, M; Braut-Boucher, F

212

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.

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

2011-01-01

213

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.

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

2012-01-01

214

The mechanism for activation of the neutrophil NADPH-oxidase by the peptides formyl-Met-Leu-Phe and Trp-Lys-Tyr-Met-Val-Met differs from that for interleukin-8  

PubMed Central

Neutrophil chemotaxis has been shown to be regulated by two different signalling pathways that allow strong chemoattractants, such as bacterial-derived formylated peptides, to dominate over endogenous attractants, such as interleukin-8 (IL-8). Here we show that triggering of the formyl peptide receptor (FPR) with f-Met-Leu-Phe (fMLF) substantially reduced the neutrophil superoxide production induced by activation of the CXC receptors with IL-8. When the order of agonists was reversed, the cells were primed in their response to fMLF, suggesting that the signalling hierarchy between strong, so-called end-type (i.e. fMLF) and weak or intermediate-type (i.e. IL-8) chemoattractants, is also operating during activation of the NADPH-oxidase. The same result was obtained when fMLF was replaced with the hexapeptide, WKYMVM, specific for the formyl peptide-like receptor 1 (FPRL1). There were additional differences between the agonist receptor pairs fMLF/FPR, WKYMVM/FPRL1 and IL-8/CXCR. In contrast to FPR and FPRL1, no reserve pool of CXCR was present in subcellular granules and it was impossible to prime the oxidative response transduced through CXCR by the addition of priming agents such as tumour necrosis factor-? and platelet-activating factor. Moreover, the cytoskeleton-disrupting substance, cytochalasin B, had no effect either on IL-8-triggered oxidase activation or on CXCR reactivation. A pertussis toxin-sensitive G-protein is involved in signalling mediated through both FPR and CXCR, and the signalling cascades include a transient intracellular calcium increase, as well as downstream p38 MAPK and phosphoinositide 3-kinase activation. The data presented in this study provide support for two different signalling pathways to the neutrophil NADPH-oxidase, used by ligand binding to FPR/FPRL1 or CXCR, respectively.

Fu, Huamei; Bylund, Johan; Karlsson, Anna; Pellme, Sara; Dahlgren, Claes

2004-01-01

215

Contrasting roles of NADPH oxidase isoforms in pressure-overload versus angiotensin II-induced cardiac hypertrophy.  

PubMed

Increased production of reactive oxygen species (ROS) is implicated in the development of left ventricular hypertrophy (LVH). Phagocyte-type NADPH oxidases are major cardiovascular sources of ROS, and recent data indicate a pivotal role of a gp91phox-containing NADPH oxidase in angiotensin II (Ang II)-induced LVH. We investigated the role of this oxidase in pressure-overload LVH. gp91phox-/- mice and matched controls underwent chronic Ang II infusion or aortic constriction. Ang II-induced increases in NADPH oxidase activity, atrial natriuretic factor (ANF) expression, and cardiac mass were inhibited in gp91phox-/- mice, whereas aortic constriction-induced increases in cardiac mass and ANF expression were not inhibited. However, aortic constriction increased cardiac NADPH oxidase activity in both gp91phox-/- and wild-type mice. Myocardial expression of an alternative gp91phox isoform, Nox4, was upregulated after aortic constriction in gp91phox-/- mice. The antioxidant, N-acetyl-cysteine, inhibited pressure-overload-induced LVH in both gp91phox-/- and wild-type mice. These data suggest a differential response of the cardiac Nox isoforms, gp91phox and Nox4, to Ang II versus pressure overload. PMID:14551238

Byrne, Jonathan A; Grieve, David J; Bendall, Jennifer K; Li, Jian-Mei; Gove, Christopher; Lambeth, J David; Cave, Alison C; Shah, Ajay M

2003-10-09

216

Short-term intravenous insulin infusion is associated with reduced expression of NADPH oxidase p47(phox) subunit in monocytes from type 2 diabetes patients.  

PubMed

Hyperglycemia is a well-known inducing factor of oxidative stress through activation of NADPH oxidase. In addition to its plasma glucose lowering effect, insulin may also have antioxidant activity and was shown to downregulate NADPH oxidase expression in vitro. In this study, we show that a short-term (3-day) intravenous insulin infusion in patients with type 2 diabetes induces normalization of both glycemia and mRNA expression of circulating monocyte p47(phox) subunit. PMID:22780425

Vaquer, Guillaume; Magous, Richard; Cros, Gérard; Wojtusciszyn, Anne; Renard, Eric; Chevassus, Hughes; Petit, Pierre; Lajoix, Anne-Dominique; Oiry, Catherine

2012-07-10

217

NITRITE CONFERS PROTECTION AGAINST MYOCARDIAL INFARCTION: ROLE OF XANTHINE OXIDOREDUCTASE, NADPH OXIDASE AND KATP CHANNELS  

PubMed Central

Reduction of nitrite to nitric oxide during ischemia protects the heart against injury from ischemia/reperfusion. However the optimal dose of nitrite and the mechanisms underlying nitrite-induced cardioprotection are not known. We determined the ability of nitrite and nitrate to confer protection against myocardial infarction in two rat models of ischemia/reperfusion injury and the role of xanthine oxidoreductase, NADPH oxidase, nitric oxide synthase and KATP channels in mediating nitrite-induced cardioprotection. In vivo and in vitro rat models of myocardial ischemia/reperfusion injury were used to cause infarction. Hearts (n=6/group) were treated with nitrite or nitrate for 15 min prior to 30 min regional ischemia and 180 min reperfusion. Xanthine oxidoreductase activity was measured after 15 min aerobic perfusion and 30 min ischemia. Nitrite reduced myocardial necrosis and decline in ventricular function following ischemia/reperfusion in the intact and isolated rat heart in a dose or concentration-dependent manner with an optimal dose of 4 mg/kg in vivo and concentration of 10 ?M in vitro. Nitrate had no effect on protection. Reduction in infarction by nitrite was abolished by inhibition of flavoprotein reductases and the molybdenum site of xanthine oxidoreductase, and was associated with an increase in activity of xanthine dehydrogenase and xanthine oxidase during ischemia. Inhibition of nitric oxide synthase had no effect on nitrite-induced cardioprotection. Inhibition of NADPH oxidase and KATP channels abolished nitrite-induced cardioprotection. Nitrite but not nitrate protects against infarction by a mechanism involving xanthine oxidoreductase, NADPH oxidase and KATP channels.

Baker, John E.; Su, Jidong; Fu, Xiangping; Hsu, Anna; Gross, Garrett J.; Tweddell, James S.; Hogg, Neil

2009-01-01

218

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

219

Positive regulation of the NADPH oxidase NOX4 promoter in vascular smooth muscle cells by E2F  

Microsoft Academic Search

The generation of reactive oxygen species (ROS) by the NOX family of NADPH oxidases is known to be involved in the regulation of many physiological cellular functions. Unlike other members of this family, NOX4 generates ROS constitutively without the need for a stimulus. The activity of NOX4 is known to be regulated, at least in part, at the level of

Li Zhang; Olivia R. Sheppard; Ajay M. Shah; Alison C. Brewer

2008-01-01

220

A prodigiosin analogue inactivates NADPH oxidase in macrophage cells by inhibiting assembly of p47phox and Rac.  

PubMed

Prodigiosins are natural red pigments that have multi-biological activities. Recently, we discovered a marine bacterial strain, which produces a red pigment. Extensive two-dimensional nuclear magnetic resonance and mass spectrometry analysis showed that the pigment is a prodigiosin analogue (PG-L-1). Here, we investigated the effect of PG-L-1 on NADPH oxidase activity in macrophage cells. PG-L-1 significantly inhibited superoxide anion (O(2)(-)) production by phorbol 12-myristate 13-acetate (PMA)-stimulated RAW 264.7 cells, a mouse macrophage cell line. The ED(50) value was estimated to be approximately 0.3 microM. PG-L-1 had no direct scavenging effect on O(2)(-) generated by hypoxanthine/xanthine oxidase system in electron spin resonance-spin trapping determinations, suggesting that this compound directly acts on the O(2)(-) production system, NADPH oxidase, in macrophage cells. We further investigated the effect of PG-L-1 on the behaviour of the cytosolic components of the NADPH oxidase, p67(phox), p47(phox), p40(phox), Rac and protein kinase C (PKC), in PMA-stimulated RAW 264.7 cells. Although PG-L-1 showed no effect on the activation of PKC, the immunoblotting analysis using specific antibodies showed that PG-L-1 strongly inhibits the association of p47(phox) and Rac in the plasma membrane of PMA-stimulated RAW 264.7 cells. These results suggest that PG-L-1 inactivates NADPH oxidase through the inhibition of the binding of p47(phox) and Rac to the membrane components of NADPH oxidase. PMID:17965429

Nakashima, Takuji; Iwashita, Takashi; Fujita, Tsuyoshi; Sato, Emiko; Niwano, Yoshimi; Kohno, Masahiro; Kuwahara, Shunsuke; Harada, Nobuyuki; Takeshita, Satoshi; Oda, Tatsuya

2007-10-27

221

INCREASED NADPH OXIDASE DERIVED SUPEROXIDE IS INVOLVED IN THE NEURONAL CELL DEATH INDUCED BY HYPOXIA ISCHEMIA IN NEONATAL HIPPOCAMPAL SLICE CULTURES  

PubMed Central

Neonatal brain hypoxia ischemia (HI) results in neuronal cell death. Previous studies indicate that reactive oxygen species (ROS) such as superoxide, play a key role in this process. However, the cellular sources have not been established. In this study we examined the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in neonatal HI brain injury and elucidated its mechanism of activation. Rat hippocampal slices were exposed to oxygen glucose deprivation (OGD) to mimic the conditions seen in HI. Initial studies confirmed an important role for NADPH oxidase derived superoxide in the oxidative stress associated with OGD. Further, the OGD-mediated increase in apoptotic cell death was inhibited by the NADPH oxidase inhibitor, apocynin. The activation of NADPH oxidase was found to be dependent on the p38 mitogen-activated protein kinase mediated phosphorylation and activation of the p47phox subunit. Using an adeno-associated virus antisense construct to selectively decrease p47phox expression in neurons, and showed that this lead to inhibition both of the increase in superoxide and neuronal cell death associated with OGD. We also found that NADPH oxidase inhibition in a neonatal rat model of HI or scavenging hydrogen peroxide (H2O2) reduced brain injury. Thus, we conclude that activation of the NADPH oxidase complex contributes to the oxidative stress during HI and that therapies targeted against this complex could exhibit neuroprotection against the brain injury associated with neonatal HI.

Lu, Qing; Wainwright, Mark S.; Harris, Valerie A.; Aggarwal, Saurabh; Hou, Yali; Rau, Thomas; Poulsen, David J.; Black, Stephen M

2012-01-01

222

Mitochondrial NADH oxidase activity of Setaria cervi.  

PubMed

The bovine filarial parasite, Setaria cervi, has been found to contain NAD(P)H oxidase activity. The system was predominantly located in the mitochondrial membranes, with very little activity available in the soluble fraction of the organelle. The membrane preparation also exhibited the presence of a reduced pyridine nucleotide transhydrogenase which converted NADPH into NADH by transferring a hydride ion. The oxidase activity was inhibited by all the respiratory inhibitors examined, with the greatest sensitivity to rotenone, a site I-specific inhibitor. The system was also found to be susceptible to exposure to anthelmintics, amongst which levamisole proved the most effective. PMID:2267725

Goyal, N; Srivastava, V M

1990-11-01

223

A Synthetic Peptide from Transforming Growth Factor-?1 Type III Receptor Inhibits NADPH Oxidase and Prevents Oxidative Stress in the Kidney of Spontaneously Hypertensive Rats.  

PubMed

Abstract Aims: The NADPH oxidases constitute a major source of superoxide anion (·O2(-)) in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by transforming growth factor-?1 (TGF-?1). We investigated whether a chronic treatment with P144, a peptide synthesized from type III TGF-?1 receptor, inhibited NADPH oxidases in the renal cortex of spontaneously hypertensive rats (SHR). Results: Here, we show that chronic administration of P144 significantly reduced the NADPH oxidase expression and activity as well as the oxidative stress observed in control vehicle-treated SHR (V-SHR). In addition, P144 was also able to reduce the significant increase in the renal fibrosis and in mRNA expression of different components of collagen metabolism, as well as in the levels of connective tissue growth factor observed in V-SHR. Finally, TGF-?1-stimulated NRK52E exhibited a significant increase in NADPH oxidase expression and activity as well as a TGF-?1-dependent intracellular pathway that were inhibited in the presence of P144. Innovation: Our experimental evidence suggests that reversing oxidative stress may be therapeutically useful in preventing fibrosis-associated renal damage. We show here that (i) the TGF-?1-NADPH oxidases axis is crucial in the development of fibrosis in an experimental hypertensive renal disease animal model, and (ii) the use of P144 reverses TGF-?1-dependent NADPH oxidase activity; thus, P144 may be considered a novel therapeutic tool in kidney disease associated with hypertension. Conclusion: We demonstrate that P144 inhibits NADPH oxidases and prevents oxidative stress in kidneys from hypertensive rats. Our data also suggest that these effects are associated with the renal antifibrotic effect of P144. Antioxid. Redox Signal. 19, 1607-1618. PMID:23350688

Baltanás, Ana; Miguel-Carrasco, José Luis; San José, Gorka; Cebrián, Carolina; Moreno, María U; Dotor, Javier; Borrás-Cuesta, Francisco; López, Begońa; González, Arantxa; Díez, Javier; Fortuńo, Ana; Zalba, Guillermo

2013-03-18

224

Regulation of cyclooxygenase-2 and cytosolic phospholipase A2 gene expression by lipopolysaccharide through the RNA-binding protein HuR: involvement of NADPH oxidase, reactive oxygen species and mitogen-activated protein kinases  

PubMed Central

BACKGROUND AND PURPOSE Lipopolysaccharide (LPS)-induced expression of cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) has been implicated in several respiratory diseases. HuR is known to enhance the expression of genes by binding to 3?-untranslated region (3?-UTR) of mRNA and stabilizing mRNA. However, the exact mechanisms by which HuR affects the stability of mRNA and modulates LPS-induced COX-2 and cPLA2 expression in human tracheal smooth muscle cells (HTSMCs) are not known. EXPERIMENTAL APPROACH The expression of prostaglandin E2 (PGE2) was measured by ELISA, and pro-inflammatory proteins were determined by use of a promoter assay, PCR or Western blot analysis. Overexpression of siRNAs to knock down the target components was used to manipulate the expression of HuR. Release of reactive oxygen species (ROS) was detected by fluorescence dye. The activation of signalling components was assessed by comparing phosphorylation levels, localization of protein kinases or coimmunoprecipitation assay. KEY RESULTS LPS induced COX-2 and cPLA2 expression via post-translational regulation of mRNA stabilization, which were attenuated by transfection with HuR siRNA in HTSMCs. In addition, LPS-stimulated NADPH oxidase activation and ROS generation were attenuated by the NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and apocynin (APO). Generation of ROS induced phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK and JNK1/2, which was attenuated by DPI and APO and the ROS scavenger N-acetylcysteine. CONCLUSIONS AND IMPLICATIONS These results suggested that in HTSMCs, LPS-induced COX-2 and cPLA2 expression is mediated through NADPH oxidase/ROS-dependent MAPKs associated with HuR accumulation in the cytoplasm. Activated MAPKs may regulate the nucleocytoplasmic shuttling of HuR, and thus induce the cytoplasmic accumulation of HuR.

Lin, Wei-Ning; Lin, Chih-Chung; Cheng, Hsin-Yi; Yang, Chuen-Mao

2011-01-01

225

Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency  

Microsoft Academic Search

Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency.BackgroundChronic renal failure (CRF) is associated with oxidative stress, the mechanism of which remains uncertain. Superoxide is the primary oxygen free radical produced in the body, NAD(P)H oxidase is the major source of superoxide production and superoxide dismutase (SOD) is responsible for removal of superoxide. We hypothesized that

Nosratola D Vaziri; Michael Dicus; Nathan D Ho; Laleh Boroujerdi-Rad; Ram K Sindhu

2003-01-01

226

NADPH oxidase participates in the oxidative damage caused by fluoride in rat spermatozoa. Protective role of ?-tocopherol.  

PubMed

Fluorosis, caused by drinking water contaminated with inorganic fluoride, is a public health problem in many areas around the world. The aim of this study was to evaluate oxidative stress in spermatozoa caused by fluoride and NADPH oxidase in relationship to fluoride. Four experimental groups of male Wistar rats were administered with deionized water, NaF, at a dose equivalent to 5?mg fluoride kg(-1) per 24?h, NaF plus 20?mg?kg(-1) per 24?h ?-tocopherol, or ?-tocopherol alone for 60 days. We evaluated several spermatozoa parameters in the four groups: standard quality analysis, superoxide dismutase (SOD) activity, the generation of reactive oxygen species (ROS), NADPH oxidase activity, TBARS formation, ultrastructural analyses of spermatozoa using transmission electron microscopy and in vitro fertilization (IVF) capacity. After 60 days of treatment, urinary excretion of fluoride was not modified by ?-tocopherol. Spermatozoa from fluoride-treated rats exhibited a significant increase in the generation of ROS, accompanied by a significant increase in NADPH oxidase activity. The increase in ROS generation was significantly diminished by diphenylene iodonium, an inhibitor of NADPH oxidase activity. In contrast, a decrease in the generation of ROS, an increase in SOD activity and the prevention of TBARS formation process were observed in spermatozoa of rats exposed to fluoride plus ?-tocopherol. Finally, ?-tocopherol treatment prevented the IVF incapacity observed in the spermatozoa from fluoride-treated rats. These results suggest that NADPH oxidase participates in the oxidative stress damage caused by subchronic exposure to fluoride. Copyright © 2010 John Wiley & Sons, Ltd. PMID:21089161

Izquierdo-Vega, Jeannett A; Sánchez-Gutiérrez, Manuel; Del Razo, Luz María

2010-11-19

227

A Phaseolus vulgaris NADPH oxidase gene is required for root infection by Rhizobia.  

PubMed

Plant NADPH oxidases [respiratory burst oxidase homologs (RBOHs)] have emerged as key players in the regulation of plant-pathogen interactions. Nonetheless, their role in mutualistic associations, such as the rhizobia-legume symbiosis, is poorly understood. In this work, nine members of the Phaseolus vulgaris Rboh gene family were identified. The transcript of one of these, PvRbohB, accumulated abundantly in shoots, roots and nodules. PvRbohB promoter activity was detected in meristematic regions of P. vulgaris roots, as well as during infection thread (IT) progression and nodule development. RNA interference (RNAi)-mediated PvRbohB down-regulation in transgenic roots reduced reactive oxygen species (ROS) production and lateral root density, and greatly impaired nodulation. Microscopy analysis revealed that progression of the ITs was impeded at the base of root hairs in PvRbohB-RNAi roots. Furthermore, the few nodules that formed in PvRbohB-down-regulated roots displayed abnormally wide ITs and reduced nitrogen fixation. These findings indicate that this common bean NADPH oxidase is crucial for successful rhizobial colonization and probably maintains proper IT growth and shape. PMID:22942250

Montiel, Jesús; Nava, Noreide; Cárdenas, Luis; Sánchez-López, Rosana; Arthikala, Manoj-Kumar; Santana, Olivia; Sánchez, Federico; Quinto, Carmen

2012-08-31

228

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

229

NADPH OXIDASE 4 MEDIATES TGF-?-INDUCED SMOOTH MUSCLE ?-ACTIN VIA p38MAPK AND SRF  

PubMed Central

In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle ?-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-? (TGF-?) as a differentiating agent, we examined Nox4-dependent signaling. TGF-? increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-?-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-?-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2 (MK-2). Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-?. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-?-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-?-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-?.

Martin-Garrido, Abel; Brown, David I.; Lyle, Alicia N.; Dikalova, Anna; Seidel-Rogol, Bonnie; Lassegue, Bernard; Martin, Alejandra San; Griendling, Kathy K.

2010-01-01

230

Neutral sphingomyelinase, NADPH oxidase and reactive oxygen species. Role in acute hypoxic pulmonary vasoconstriction.  

PubMed

The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ? (PKC?) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium-denuded PA segments and their inhibition prevented hypoxia-induced voltage-gated potassium channel (K(V) ) inhibition and pulmonary vasoconstriction. Consistently, H(2) O(2) , or its analogue t-BHP, decreased K(V) currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia-induced ROS production, K(V) channel inhibition and vasoconstriction. Hypoxia induced p47(phox) phosphorylation and its interaction with caveolin-1. Inhibition of nSMase (GW4869) or PKC? prevented p47(phox) phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKC? sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase-PKC?-NADPH oxidase as a necessary step required for ROS production and vasoconstriction. PMID:21792922

Frazziano, Giovanna; Moreno, Laura; Moral-Sanz, Javier; Menendez, Carmen; Escolano, Lucía; Gonzalez, Constancio; Villamor, Eduardo; Alvarez-Sala, Jose Luis; Cogolludo, Angel L; Perez-Vizcaino, Francisco

2011-10-01

231

Vascular NAD(P)H oxidases: specific features, expression, and regulation.  

PubMed

The importance of reactive oxygen species (ROS) in vascular physiology and pathology is becoming increasingly evident. All cell types in the vascular wall produce ROS derived from superoxide-generating protein complexes similar to the leukocyte NADPH oxidase. Specific features of the vascular enzymes include constitutive and inducible activities, substrate specificity, and intracellular superoxide production. Most phagocyte enzyme subunits are found in vascular cells, including the catalytic gp91phox (aka, nox2), which was the earliest member of the newly discovered nox family. However, smooth muscle frequently expresses nox1 rather than gp91phox, and nox4 is additionally present in all cell types. In cell culture, agonists increase ROS production by activating multiple signals, including protein kinase C and Rac, and by upregulating oxidase subunits. The oxidases are also upregulated in vascular disease and are involved in the development of atherosclerosis and a significant part of angiotensin II-induced hypertension, possibly via nox1 and nox4. Likewise, enhanced vascular oxidase activity is associated with diabetes. Therefore, members of this enzyme family appear to be important in vascular biology and disease and constitute promising targets for future therapeutic interventions. PMID:12855411

Lassčgue, Bernard; Clempus, Roza E

2003-08-01

232

NO-mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells.  

PubMed

The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (.O(2)(-)) formation. In this study, we analysed the effect of laminar shear stress on .O(2)(-) formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone-and-plate viscometer for up to 24 h. Short-term application of shear stress transiently induced .O(2)(-) formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds-tat, but NAD(P)H oxidase subunit expression was unchanged. Long-term arterial laminar shear stress (30 dyne cm(-2), 24 h) down-regulated .O(2)(-) formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91(phox) and p47(phox). In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down-regulation of .O(2)(-) formation, gp91(phox) and p47(phox) expression by long-term laminar shear stress was blocked by l-NAME. NO donor DETA-NO down-regulates .O(2)(-) formation, gp91(phox) and p47(phox) expression in static cultures. In conclusion, our data suggest a transient activation of .O(2)(-) formation by short-term shear stress, followed by a down-regulation of endothelial NAD(P)H oxidase in response to long-term laminar shear stress. NO-mediated down-regulation by shear stress preferentially affects the gp91(phox)/p47(phox)-containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/.O(2)(-) balance and the vasoprotective potential of physiological levels of laminar shear stress. PMID:16873416

Duerrschmidt, Nicole; Stielow, Claudia; Muller, Gregor; Pagano, Patrick J; Morawietz, Henning

2006-07-27

233

Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox\\/Duox) family of enzymes  

Microsoft Academic Search

BACKGROUND: NADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox\\/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.

Tsukasa Kawahara; Mark T Quinn; J David Lambeth

2007-01-01

234

Oxidative stress and endothelial dysfunction in aortas of aged spontaneously hypertensive rats by NOX1/2 is reversed by NADPH oxidase inhibition.  

PubMed

Arterial hypertension is associated with increased levels of reactive oxygen species, which may scavenge endothelium-derived NO and thereby diminish its vasorelaxant effects. However, the quantitatively relevant source of reactive oxygen species is unclear. Thus, this potential pathomechanism is not yet pharmacologically targetable. Several enzymatic sources of reactive oxygen species have been suggested: uncoupled endothelial NO synthase, xanthine oxidase, and NADPH oxidases. Here we show that increased reactive oxygen species formation in aortas of 12- to 14-month-old spontaneously hypertensive rats versus age-matched Wistar Kyoto rats is inhibited by the specific NADPH oxidase inhibitor VAS2870 but neither by the xanthine oxidase inhibitor oxypurinol nor the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester. NADPH oxidase activity, as well as protein expression of its catalytic subunits, NOX1 and NOX2, was increased in the aortas of spontaneously hypertensive rats, whereas the expression of NOX4 protein, the most abundant NOX isoform, was not significantly changed. Impaired acetylcholine-induced relaxation of spontaneously hypertensive rat aortas was significantly improved by VAS2870. In conclusion, NOX1 and NOX2 but not NOX4 proteins are increased in aged spontaneously hypertensive rat aortas. Importantly, these NOX isoforms, in particular, ectopic expression of NOX1 in endothelial cells, appear to affect vascular function in an NADPH oxidase inhibitor-reversible manner. NADPH oxidases may, thus, be a novel target for the treatment of systemic hypertension. PMID:20606112

Wind, Sven; Beuerlein, Knut; Armitage, Melanie E; Taye, Ashraf; Kumar, Arun H S; Janowitz, Daniel; Neff, Christina; Shah, Ajay M; Wingler, Kirstin; Schmidt, Harald H H W

2010-07-06

235

Pivotal Role of a gp91phox-Containing NADPH Oxidase in Angiotensin II-Induced Cardiac Hypertrophy in Mice  

Microsoft Academic Search

Background—Angiotensin II induces both cardiac and vascular smooth muscle (VSM) hypertrophy. Recent studies suggest a central role for a phagocyte-type NADPH oxidase in angiotensin II-induced VSM hypertrophy. The possible involvement of an NADPH oxidase in the development of cardiac hypertrophy has not been studied. Methods and Results—Mice with targeted disruption of the NADPH oxidase subunit gp91phox (gp91phox\\/) and matched wild-type

Jennifer K. Bendall; Alison C. Cave; Christophe Heymes; Nicholas Gall; Ajay M. Shah

236

The Nonphagocytic NADPH Oxidase Duox1 Mediates a Positive Feedback Loop During T Cell Receptor Signaling  

PubMed Central

Production of reactive oxygen species, often by NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidases, plays a role in the signaling responses of cells to many receptor stimuli. Here, we describe the function of the calcium-dependent, nonphagocytic NADPH oxidase Duox1 in primary human CD4+ T cells and cultured T cell lines. Duox1 bound to inositol 1,4,5-trisphosphate receptor 1 and was required for early T cell receptor (TCR)–stimulated production of hydrogen peroxide (H2O2) through a pathway that was dependent on TCR-proximal kinases. Transient or stable knockdown of Duox1 inhibited TCR signaling, especially phosphorylation of tyrosine-319 of ? chain–associated protein kinase of 70 kilodaltons (ZAP-70), store-operated entry of calcium ions (Ca2+), and activation of extracellular signal–regulated kinase. The production of cytokines was also inhibited by knockdown of Duox1. Duox1-mediated inactivation of Src homology 2 domain–containing protein tyrosine phosphatase 2 promoted the phosphorylation of ZAP-70 and its association with the Src family tyrosine kinase Lck and the CD3? chain of the TCR complex. Thus, we suggest that activation of Duox1, downstream of proximal TCR signals, generates H2O2 that acts in a positive feedback loop to enhance and sustain further TCR signaling.

Kwon, Jaeyul; Shatynski, Kristen E.; Chen, Haiyan; Morand, Stanislas; de Deken, Xavier; Miot, Francoise; Leto, Thomas L.; Williams, Mark S.

2010-01-01

237

The Importance of Myeloperoxidase in Apocynin-Mediated NADPH Oxidase Inhibition  

PubMed Central

Apocynin is widely used as an inhibitor of the NADPH oxidase. Since myeloperoxidase (MPO) has been considered as essential for the mechanism of action of apocynin, here we used cells with different levels of MPO and compared their sensitivity to apocynin. HL-60 cells were differentiated with DMSO or IFN?/TNF? and compared with peripheral mononuclear (PBMC) and polymorphonuclear cells (PMN). The relative MPO activity was PBMC = HL60 DMSO < HL60 IFN? < PMN. Apocynin inhibited the intracellular reactive oxygen species production by PMN (80%) and IFN?/TNF?-differentiated HL-60 cells (45%) but showed a minor effect in PBMC and DMSO differentiated HL-60 cells (20%). The addition of azide decreased the efficiency of apocynin in PMN and the addition of peroxidase increased the inhibition in PBMC. We also determined the gene expression of the components gp91phox, p47phox, p22phox and p67phox in the resting cells. Apocynin did not change gp91phox, p47phox or p22phox gene expression in nonstimulated PBMC, HL60 DMSO, HL60 IFN?/TNF?, and PMN and has a subtle increase in p67phox in HL60 IFN?/TNF?. The results from this work suggest that a rational search for better inhibitors of NADPH oxidase in leukocytes should include a correlation with their affinity as substrates for MPO.

de Almeida, Ana Carolina; dos Santos Vilela, Maria Marluce; Condino-Neto, Antonio; Ximenes, Valdecir F.

2012-01-01

238

Peroxisome Proliferator-Activated Receptor Induces NADPH Oxidase Activity in Macrophages, Leading to the Generation of LDL with PPAR Activation Properties  

Microsoft Academic Search

Abstract—Peroxisome,proliferator–activated receptors (PPARs) are nuclear receptors controlling lipid and glucose metabolism as well as inflammation. PPARs are expressed in macrophages, cells that also generate reactive oxygen species (ROS). In this study, we investigated whether PPARs regulate ROS production in macrophages. Different PPAR-, but not PPAR- agonists, increased the production of ROS (H2O2 and O2 .) in human and murine macrophages.

Elisabeth Teissier; Atsushi Nohara; Giulia Chinetti; Rejane Paumelle; Bertrand Cariou; Jean-Charles Fruchart; Ralf P. Brandes; Ajay Shah; Bart Staels

2004-01-01

239

NADPH oxidases, reactive oxygen species, and hypertension: clinical implications and therapeutic possibilities.  

PubMed

Reactive oxygen species (ROS) influence many physiological processes including host defense, hormone biosynthesis, fertilization, and cellular signaling. Increased ROS production (termed "oxidative stress") has been implicated in various pathologies, including hypertension, atherosclerosis, diabetes, and chronic kidney disease. A major source for vascular and renal ROS is a family of nonphagocytic NAD(P)H oxidases, including the prototypic Nox2 homolog-based NAD(P)H oxidase, as well as other NAD(P)H oxidases, such as Nox1 and Nox4. Other possible sources include mitochondrial electron transport enzymes, xanthine oxidase, cyclooxygenase, lipoxygenase, and uncoupled nitric oxide synthase. NAD(P)H oxidase-derived ROS plays a physiological role in the regulation of endothelial function and vascular tone and a pathophysiological role in endothelial dysfunction, inflammation, hypertrophy, apoptosis, migration, fibrosis, angiogenesis, and rarefaction, important processes underlying cardiovascular and renal remodeling in hypertension and diabetes. These findings have evoked considerable interest because of the possibilities that therapies against nonphagocytic NAD(P)H oxidase to decrease ROS generation and/or strategies to increase nitric oxide (NO) availability and antioxidants may be useful in minimizing vascular injury and renal dysfunction and thereby prevent or regress target organ damage associated with hypertension and diabetes. Here we highlight current developments in the field of reactive oxygen species and cardiovascular disease, focusing specifically on the recently identified novel Nox family of NAD(P)H oxidases in hypertension. We also discuss the potential role of targeting ROS as a therapeutic possibility in the management of hypertension and cardiovascular disease. PMID:18227481

Paravicini, Tamara M; Touyz, Rhian M

2008-02-01

240

NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality  

PubMed Central

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.

Brothers, Kimberly M.; Gratacap, Remi L.; Barker, Sarah E.; Newman, Zachary R.; Norum, Ashley; Wheeler, Robert T.

2013-01-01

241

NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality.  

PubMed

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis. PMID:24098114

Brothers, Kimberly M; Gratacap, Remi L; Barker, Sarah E; Newman, Zachary R; Norum, Ashley; Wheeler, Robert T

2013-10-03

242

Non-steroidal anti-inflammatory drugs activate NADPH oxidase in adipocytes and raise the H2O2 pool to prevent cAMP-stimulated protein kinase a activation and inhibit lipolysis  

PubMed Central

Background Non-steroidal anti-inflammatory drugs (NSAIDs) —aspirin, naproxen, nimesulide, and piroxicam— lowered activation of type II cAMP-dependent protein kinase A (PKA-II) in isolated rat adipocytes, decreasing adrenaline- and dibutyryl cAMP (Bt2cAMP)-stimulated lipolysis. The molecular bases of insulin-like actions of NSAID were studied. Results Based on the reported inhibition of lipolysis by H2O2, catalase was successfully used to block NSAID inhibitory action on Bt2cAMP-stimulated lipolysis. NSAID, at (sub)micromolar range, induced an H2O2 burst in rat adipocyte plasma membranes and in whole adipocytes. NSAID-mediated rise of H2O2 was abrogated in adipocyte plasma membranes by: diphenyleneiodonium, an inhibitor of NADPH oxidase (NOX); the NOX4 antibody; and cytochrome c, trapping the NOX-formed superoxide. These three compounds prevented the inhibition of Bt2cAMP-stimulated lipolysis by NSAIDs. Inhibition of aquaporin-mediated H2O2 transport with AgNO3 in adipocytes allowed NOX activation but prevented the lipolysis inhibition promoted by NSAID: i.e., once synthesized, H2O2 must reach the lipolytic machinery. Since insulin inhibits adrenaline-stimulated lipolysis, the effect of aspirin on isoproterenol-stimulated lipolysis in rat adipocytes was studied. As expected, isoproterenol-mediated lipolysis was blunted by both insulin and aspirin. Conclusions NSAIDs activate NOX4 in adipocytes to produce H2O2, which impairs cAMP-dependent PKA-II activation, thus preventing isoproterenol-activated lipolysis. H2O2 signaling in adipocytes is a novel and important cyclooxygenase-independent effect of NSAID.

2013-01-01

243

Neuronal expression of the NADPH oxidase NOX4, and its regulation in mouse experimental brain ischemia  

Microsoft Academic Search

Ischemia-induced neuronal damage has been linked to elevated production of reactive oxygen species (ROS) both in animal models and in humans. NADPH oxidase enzymes (NOX-es) are a major enzymatic source of ROS, but their role in brain ischemia has not yet been investigated. The present study was carried out to examine the expression of NOX4, one of the new NADPH

P. Vallet; Y. Charnay; K. Steger; E. Ogier-Denis; E. Kovari; F. Herrmann; J.-P. Michel; I. Szanto

2005-01-01

244

Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney.  

PubMed

Phagocytes generate superoxide anion (O(2)(-)) by a classic, 5-component NADPH oxidase. O(2)(-) contributes to hypertension in spontaneously hypertensive rats (SHR). Therefore, we tested the hypothesis that NADPH oxidase expression is enhanced in the SHR kidney. We also analyzed the localization of NADPH oxidase components in SHR kidney. Renal NADPH oxidase was quantified by reverse transcription-polymerase chain reaction and Western blotting and was localized in SHR and Wistar Kyoto rat (WKY) kidney by immunohistochemistry. The mRNA for 5 subunits of phagocyte NADPH oxidase, and also for MOX1 and RENOX (NOX4), was detected in adult rat kidney. Kidneys of adult (10 weeks old) SHR had a significantly (P<0.01) greater mRNA for p47phox (SHR 0.81 +/- 0.05 versus WKY 0.37 +/- 0.01, arbitrary unit), which was confirmed by Western blotting (SHR 0.58 +/- 0.04 versus WKY 0.42 +/- 0.04, arbitrary unit; P<0.05) and by immunohistochemistry. This higher p47phox protein expression was also detected in young prehypertensive SHR (SHR 0.61 +/- 0.05 versus WKY 0.39 +/- 0.04, arbitrary unit; P<0.01). The 10-week-old SHR contained more modest but significantly (P<0.05) greater protein for p67phox (SHR 0.54 +/- 0.02 versus WKY 0.46 +/- 0.02). Immunostaining localized p47phox, p67phox, and p22phox in vasculature, macula densa, distal convoluted tubule, cortical collecting duct, and outer and inner medullary collecting ducts. The kidney of SHR expresses genes for all the main components of phagocyte NADPH oxidase, RENOX, and MOX1. There is a prominent increase in the SHR kidney of the mRNA, and protein expression of p47phox in the vasculature, macula densa, and distal nephron, which precedes development of hypertension. PMID:11847196

Chabrashvili, Tinatin; Tojo, Akahiro; Onozato, Maristela Lika; Kitiyakara, Chagriya; Quinn, Mark T; Fujita, Toshiro; Welch, William J; Wilcox, Christopher S

2002-02-01

245

Regional expression of NO synthase, NAD(P)H oxidase and superoxide dismutase in the rat brain  

Microsoft Academic Search

Nitric oxide (NO) derived from the endothelial NO synthase (eNOS) contributes to regulation of cerebral circulation, whereas that produced by neuronal NOS (nNOS) participates in the regulation of brain function. In particular, NO plays an important role in modulation of sympathetic activity and hence central regulation of arterial pressure. Superoxide derived from NAD(P)H oxidase avidly reacts with and inactivates NO

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

2007-01-01

246

Role of NADPH Oxidase in the Vascular Hypertrophic and Oxidative Stress Response to Angiotensin II in Mice  

Microsoft Academic Search

Oxygen-derived free radicals are involved in the vascular response to angiotensin II (Ang II), but the role of NADPH oxidase, its subunit proteins, and their vascular localization remain controversial. Our purpose was to address the role of NADPH oxidase in the blood pressure (BP), aortic hypertrophic, and oxidant responses to Ang II by taking advantage of knockout (KO) mice that

Hui Di Wang; Shanqin Xu; Douglas G. Johns; Yue Du; Mark T. Quinn; Antonio J. Cayatte; Richard A. Cohen

247

Selective Detection of NADPH Oxidase in Polymorphonuclear Cells by Means of NAD(P)H-Based Fluorescence Lifetime Imaging  

PubMed Central

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670±140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function.

Niesner, R.; Narang, P.; Spiecker, H.; Andresen, V.; Gericke, K. -H.; Gunzer, M.

2008-01-01

248

Selective detection of NADPH oxidase in polymorphonuclear cells by means of NAD(P)H-based fluorescence lifetime imaging.  

PubMed

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670 +/- 140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function. PMID:20107577

Niesner, R; Narang, P; Spiecker, H; Andresen, V; Gericke, K-H; Gunzer, M

2008-11-16

249

Characterization of bovine endothelial nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of haem in dimerization.  

PubMed Central

The fatty-acylation-deficient bovine endothelial NO synthase (eNOS) mutant (Gly-2 to Ala-2, G2AeNOS) was purified from a baculovirus overexpression system. The purified protein was soluble and highly active (0.2-0.7 micromol of l-citrulline. mg-1.min-1), contained 0. 77+/-0.01 equivalent of haem per subunit, showed a Soret maximum at 396 nm, and exhibited only minor uncoupling of NADPH oxidation in the absence of l-arginine or tetrahydrobiopterin. Radioligand binding studies revealed KD values of 147+/-24.1 nM and 52+/-9.2 nM for specific binding of tetrahydrobiopterin in the absence and presence of 0.1 mM l-arginine respectively. The positive co-operative effect of l-arginine was due to a pronounced decrease in the rate of tetrahydrobiopterin dissociation (from 1.6+/-0.5 to 0. 3+/-0.1 min-1). Low-temperature SDS gel electrophoresis showed that approx. 80% of the protein migrated as haem-containing dimer after preincubation with l-arginine and tetrahydrobiopterin. Gel-filtration chromatography yielded one peak with a Stokes radius of 6.8+/-0.04 nm, corresponding to a hydrodynamic volume of 1. 32x10(-24) m3, whereas haem-deficient preparations (approx. 0.3 equivalent per subunit) contained an additional protein species with a hydrodynamic radius of 5.1+/-0.2 nm and a corresponding volume of 0.55x10(-24) m3, suggesting that haem availability regulates eNOS dimerization.

List, B M; Klosch, B; Volker, C; Gorren, A C; Sessa, W C; Werner, E R; Kukovetz, W R; Schmidt, K; Mayer, B

1997-01-01

250

Oscillatory Shear Stress Induces Mitochondrial Superoxide Production: Implication of NADPH Oxidase and c-Jun NH2-Terminal Kinase Signaling  

PubMed Central

Abstract Fluid shear stress is intimately linked with vascular oxidative stress and atherosclerosis. We posited that atherogenic oscillatory shear stress (OSS) induced mitochondrial superoxide (mtO2•?) production via NADPH oxidase and c-Jun NH2-terminal kinase (JNK-1 and JNK-2) signaling. In bovine aortic endothelial cells, OSS (±3?dyn/cm2) induced JNK activation, which peaked at 1?h, accompanied by an increase in fluorescein isothiocyanate-conjugated JNK fluorescent and MitoSOX Red (specific for mtO2•? production) intensities. Pretreatment with apocynin (NADPH oxidase inhibitor) or N-acetyl cysteine (antioxidant) significantly attenuated OSS-induced JNK activation. Apocynin further reduced OSS-mediated dihydroethidium and MitoSOX Red intensities specific for cytosolic O2•? and mtO2•? production, respectively. As a corollary, transfecting bovine aortic endothelial cells with JNK siRNA (siJNK) and pretreating with SP600125 (JNK inhibitor) significantly attenuated OSS-mediated mtO2•? production. Immunohistochemistry on explants of human coronary arteries further revealed prominent phosphorylated JNK staining in OSS-exposed regions. These findings indicate that OSS induces mtO2•? production via NADPH oxidase and JNK activation relevant for vascular oxidative stress. Antioxid. Redox Signal. 15, 1379–1388.

Takabe, Wakako; Jen, Nelson; Ai, Lisong; Hamilton, Ryan; Wang, Sky; Holmes, Kristin; Dharbandi, Farhad; Khalsa, Bhavraj; Bressler, Steven; Barr, Mark L.; Li, Rongsong

2011-01-01

251

Oscillatory shear stress induces mitochondrial superoxide production: implication of NADPH oxidase and c-Jun NH2-terminal kinase signaling.  

PubMed

Fluid shear stress is intimately linked with vascular oxidative stress and atherosclerosis. We posited that atherogenic oscillatory shear stress (OSS) induced mitochondrial superoxide (mtO2•-) production via NADPH oxidase and c-Jun NH(2)-terminal kinase (JNK-1 and JNK-2) signaling. In bovine aortic endothelial cells, OSS (±3?dyn/cm2) induced JNK activation, which peaked at 1?h, accompanied by an increase in fluorescein isothiocyanate-conjugated JNK fluorescent and MitoSOX Red (specific for mtO2•- production) intensities. Pretreatment with apocynin (NADPH oxidase inhibitor) or N-acetyl cysteine (antioxidant) significantly attenuated OSS-induced JNK activation. Apocynin further reduced OSS-mediated dihydroethidium and MitoSOX Red intensities specific for cytosolic O2•- and mtO2•- production, respectively. As a corollary, transfecting bovine aortic endothelial cells with JNK siRNA (siJNK) and pretreating with SP600125 (JNK inhibitor) significantly attenuated OSS-mediated mtO2•- production. Immunohistochemistry on explants of human coronary arteries further revealed prominent phosphorylated JNK staining in OSS-exposed regions. These findings indicate that OSS induces mtO2•- production via NADPH oxidase and JNK activation relevant for vascular oxidative stress. PMID:20919940

Takabe, Wakako; Jen, Nelson; Ai, Lisong; Hamilton, Ryan; Wang, Sky; Holmes, Kristin; Dharbandi, Farhad; Khalsa, Bhavraj; Bressler, Steven; Barr, Mark L; Li, Rongsong; Hsiai, Tzung K

2011-04-14

252

Inhibition of NADPH Oxidase Mediates Protective Effect of Cardiotonic Pills against Rat Heart Ischemia/Reperfusion Injury  

PubMed Central

Cardiotonic pill (CP) is a compound Chinese medicine currently used in China for treatment of ischemic angina pectoris. Our previous results indicated that a single dosing of CP pretreatment at 0.8?g/kg attenuates ischemia/reperfusion- (I/R-) induced myocardial injury and cardiac microcirculatory disturbance. The present study aimed to investigate the effect of CP at low dosage in a multiple dosing manner and to uncover the mechanism of antioxidative activity of CP. Male Sprague-Dawley rats were subjected to left anterior descending artery occlusion for 30?min followed by 60?min reperfusion. CP was administrated daily by gavage for six days at 0.1, 0.4, and 0.8?g/kg/day before I/R. Results showed that multiple dosing of CP at three doses significantly reduced I/R-induced myocardial injury, microcirculatory disturbance, and oxidative stress. CP dramatically inhibited I/R-induced nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase subunit gp91phox expression and p67phox and p47phox translocation from cytosol to cell membrane. Translocation of cytosolic subunits to membrane is required for the activation of NADPH oxidase. These data suggested that multiple dosing of CP at doses ranging from 0.1 to 0.8?g/kg/day reduced I/R-induced rat myocardial injury and microcirculatory disturbance, which was mediated by inhibition of NADPH oxidase activation.

Yang, Xiao-Yuan; Zhao, Na; Liu, Yu-Ying; Hu, Bai-He; Sun, Kai; Chang, Xin; Wei, Xiao-Hong; Fan, Jing-Yu; Han, Jing-Yan

2013-01-01

253

Two NADPH oxidase isoforms are required for sexual reproduction and ascospore germination in the filamentous fungus Podospora anserina  

Microsoft Academic Search

NADPH oxidases are enzymes that produce reactive oxygen species (ROS) using electrons derived from intracellular NADPH. In plants and mammals, ROS have been proposed to be second messengers that signal defence responses or cell proliferation. By inactivating PaNox1 and PaNox2, two genes encoding NADPH oxidases, we demonstrate the crucial role of these enzymes in the control of two key steps

Fabienne Malagnac; Hervé Lalucque; Gersende Lepčre; Philippe Silar

2004-01-01

254

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

255

Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4.  

PubMed

NADPH oxidase Nox4 is expressed in a wide range of tissues and plays a role in cellular signaling by providing reactive oxygen species (ROS) as intracellular messengers. Nox4 oxidase activity is thought to be constitutive and regulated at the transcriptional level; however, we challenge this point of view and suggest that specific quinone derivatives could modulate this activity. In fact, we demonstrated a significant stimulation of Nox4 activity by 4 quinone derivatives (AA-861, tBuBHQ, tBuBQ, and duroquinone) observed in 3 different cellular models, HEK293E, T-REx™, and chondrocyte cell lines. Our results indicate that the effect is specific toward Nox4 versus Nox2. Furthermore, we showed that NAD(P)H:quinone oxidoreductase (NQO1) may participate in this stimulation. Interestingly, Nox4 activity is also stimulated by reducing agents that possibly act by reducing the disulfide bridge (Cys226, Cys270) located in the extracellular E-loop of Nox4. Such model of Nox4 activity regulation could provide new insight into the understanding of the molecular mechanism of the electron transfer through the enzyme, i.e., its potential redox regulation, and could also define new therapeutic targets in diseases in which quinones and Nox4 are implicated. PMID:23583257

Nguyen, Minh Vu Chuong; Lardy, Bernard; Rousset, Francis; Hazane-Puch, Florence; Zhang, Leilei; Trocmé, Candice; Serrander, Lena; Krause, Karl-Heinz; Morel, Françoise

2013-04-09

256

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.

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

257

Quercetin inhibits vascular superoxide production induced by endothelin-1: Role of NADPH oxidase, uncoupled eNOS and PKC.  

PubMed

Chronic administration of the most abundant dietary flavonoid quercetin exerts antihypertensive effects and improves endothelial function. We have investigated the effects of quercetin and its methylated metabolite isorhamnetin (1-10microM) on endothelial dysfunction and superoxide (O(2*)(-)) production induced by endothelin-1 (ET-1, 10nM). ET-1 increased the contractile response induced by phenylephrine and reduced the relaxant responses to acetylcholine in phenylephrine contracted intact aorta, and these effects were prevented by co-incubation with quercetin, isorhamnetin or chelerythrine (protein kinase C (PKC) inhibitor). This endothelial dysfunction was also improved by superoxide dismutase (SOD), apocynin (NADPH oxidase inhibitor) and sepiapterin (tetrahydrobiopterin synthesis substrate). Furthermore, ET-1 increased intracellular O(2*)(-) production in all layers of the vessel, protein expression of NADPH oxidase subunit p47(phox) without affecting p22(phox) expression and lucigenin-enhanced chemiluminescence signal stimulated by calcium ionophore A23187. All these changes were prevented by both quercetin and isorhamnetin. Moreover, apocynin, endothelium denudation and N(G)-nitro-l-arginine methylester (l-NAME, nitric oxide synthase inhibitor) suppressed the ET-1-induced increase in A23187-stimulated O(2*)(-) generation. Moreover, quercetin but not isorhamnetin, inhibited the increased PKC activity induced by ET-1. Taken together these results indicate that ET-1-induced NADPH oxidase up-regulation and eNOS uncoupling via PKC leading to endothelial dysfunction and these effects were prevented by quercetin and isorhamnetin. PMID:18436224

Romero, Miguel; Jiménez, Rosario; Sánchez, Manuel; López-Sepúlveda, Rocío; Zarzuelo, Maria José; O'Valle, Francisco; Zarzuelo, Antonio; Pérez-Vizcaíno, Francisco; Duarte, Juan

2008-03-16

258

Myofibroblast Differentiation During Fibrosis: Role of NAD(P)H Oxidases  

PubMed Central

Progression of fibrosis involves interstitial hypercellularity, matrix accumulation and atrophy of epithelial structures, resulting in the loss of normal function and ultimately organ failure. There is common agreement that the fibroblast/myofibroblast is the cell type most responsible for interstitial matrix accumulation and consequent structural deformations associated with fibrosis. During wound healing and progressive fibrotic events fibroblasts transform into myofibroblasts acquiring smooth muscle features, most notably the expression of alpha-smooth muscle actin (?-SMA) and synthesis of mesenchymal cell related matrix proteins. In renal disease, glomerular mesangial cells also acquire a myofibroblast phenotype and synthesize the same matrix proteins. The origin of interstitial myofibroblasts during fibrosis is a matter of debate where the cells are proposed to derive from resident fibroblasts, pericytes, perivascular adventitial, epithelial, and/or endothelial sources. Regardless of the origin of the cells, TGF-?1 is the principal growth factor responsible for myofibroblast differentiation to a profibrotic phenotype and exerts its effects via Smad signaling pathways involving MAPK and Akt/PKB. Additionally, reactive oxygen species (ROS) play important roles in progression of fibrosis. ROS are derived from a variety of enzyme sources of which the NAD(P)H oxidase family has been identified as a major source of superoxide and hydrogen peroxide generation in the cardiovasculature and the kidney during health and disease. Recent evidence indicates that the NAD(P)H oxidase homolog Nox4 is most accountable for ROS-induced fibroblast and mesangial cell activation where it has an essential role in TGF-?1 signaling of fibroblast activation and differentiation into a profibrotic myofibroblast phenotype and matrix production. Information on the role of ROS in mesangial cell and fibroblast signaling is incomplete and further research on myofibroblast differentiation during fibrosis is warranted.

Barnes, Jeffrey L.; Gorin, Yves

2013-01-01

259

Delayed association of the NADPH oxidase complex with macrophage vacuoles containing the opportunistic pathogen Burkholderia cenocepacia  

Microsoft Academic Search

Burkholderia cenocepacia causes chronic lung infections in patients suffering from cystic fibrosis and chronic granulomatous disease. We have previously shown that B. cenocepacia survives intracellularly in macrophages within a membrane vacuole (BcCV) that delays acidification. Here, we report that after macrophage infection with live B. cenocepacia there is a ~6 h delay in the association of NADPH oxidase with BcCVs,

Karen E. Keith; Daniel W. Hynes; Judith E. Sholdice; Miguel A. Valvano

2009-01-01

260

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation  

Microsoft Academic Search

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we

Mariapaola Nitti; Anna Lisa Furfaro; Claudia Cevasco; Nicola Traverso; Umberto Maria Marinari; Maria Adelaide Pronzato; Cinzia Domenicotti

2010-01-01

261

The Role of Adrenomedullin in the Renal NADPH Oxidase and (Pro)renin in Diabetic Mice  

PubMed Central

Adrenomedullin has an antioxidative action and protects organs in various diseases. To clarify the role of adrenomedullin in diabetic nephropathy, we investigated the NADPH oxidase expression, renin-secreting granular cell (GC) hyperplasia, and glomerular matrix expansion in the streptozotocin (STZ)-induced diabetic adrenomedullin gene knockout (AMKO) mice compared with the STZ-diabetic wild mice at 10 weeks. The NADPH oxidase p47phox expression and lipid peroxidation products were enhanced in the glomeruli of the diabetic mice compared with that observed in the controls in both wild and AMKO mice. These changes were more obvious in the AMKO mice than in the wild mice. Glomerular mesangial matrix expansion was more severe in the diabetic AMKO mice than in the diabetic wild mice and exhibited a positive correlation with the degree of lipid peroxidation products in the glomeruli. Proteinuria was significantly higher in the diabetic AMKO mice than in the diabetic wild mice. The GC hyperplasia score and the renal prorenin expression were significantly increased in the diabetic AMKO mice than in the diabetic wild mice, and a positive correlation was observed with the NADPH oxidase expression in the macula densa. The endogenous adrenomedullin gene exhibits an antioxidant action via the inhibition of NADPH oxidase probably by suppressing the local renin-angiotensin system.

Hayashi, Michio; Fujita, Toshiro

2013-01-01

262

Apocynin-Treatment Reverses Hyperoxaluria Induced Changes in NADPH Oxidase System Expression in Rat Kidneys: A Transcriptional Study  

PubMed Central

Purpose We have previously shown that production of reactive oxygen species (ROS) is an important contributor to renal injury and inflammation following exposure to oxalate (Ox) or calcium-oxalate (CaOx) crystals. The present study was conducted, utilizing global transcriptome analyses, to determine the effect of Apocynin on changes in the NADPH oxidase system activated in kidneys of rats fed a diet leading to hyperoxaluria and CaOx crystal deposition. Approach Age-, sex- and weight-matched rats were either fed regular rat chow or regular rat chow supplemented with 5% w/w hydroxy-L-proline (HLP). Half of the rats on the HLP diet were also placed on Apocynin-supplemented H2O. After 28 days, each rat was euthanized, their kidneys freshly explanted and dissected to obtain both cortex and medulla tissues. Total RNA was extracted from each tissue and subjected to genomic microarrays to obtain global transcriptome data. KEGG was used to identify gene clusters with differentially expressed genes. Immunohistochemistry was used to confirm protein expressions of selected genes. Results Genes encoding both membrane- and cytosolic-NADPH oxidase complex-associated proteins, together with p21rac and Rap1a, were coordinately up-regulated significantly in both renal medulla and cortex tissues in the HLP-fed rats compared to normal healthy untreated controls. Activation of NADPH oxidase appears to occur via the angiotensin-II/angiotensin-II receptor-2 pathway, although the DAG-PKC pathway of neutrophils may also contribute. Immuno histochemical staining confirmed up-regulated gene expressions. Simultaneously, genes encoding ROS scavenger proteins were down-regulated. HLP-fed rats receiving Apocynin had a complete reversal in the differential-expression of the NADPH oxidase system genes, despite showing similar levels of hyperoxaluria. Conclusions A strong up-regulation of an oxidative/respiratory burst involving the NADPH oxidase system, activated via the angiotensin-II and most likely the DAG-PKC pathways, occurs in kidneys of hyperoxaluric rats. Apocynin treatment reversed this activation without affecting the levels of hyperoxaluria.

Joshi, Sunil; Saylor, Benjamin T.; Wang, Wei; Peck, Ammon B.; Khan, Saeed R.

2012-01-01

263

Current molecular models for NADPH oxidase regulation by Rac GTPase  

Microsoft Academic Search

Reactive oxygen species (ROS) have be en increasingly recognized as important components of cell signaling, in addition to their well- established roles in host defense. The formation of ROS in phagocytic - and nonphagocytic cells involves membrane - localized and Rac GTPase-regulated NADPH oxida se(s). We discuss here the current molecular models for Rac GTPase action in the control of

Gary M. Bokoch; Becky A. Diebold

2002-01-01

264

Pyocyanin-Enhanced Neutrophil Extracellular Trap Formation Requires the NADPH Oxidase  

PubMed Central

Beyond intracellular killing, a novel neutrophil-based antimicrobial mechanism has been recently discovered: entrapment and killing by neutrophil extracellular traps (NETs). NETs consist of extruded nuclear DNA webs decorated with granule proteins. Although NET formation is an important innate immune mechanism, uncontrolled NET release damages host tissues and has been linked to several diseases including cystic fibrosis (CF). The major CF airway pathogen Pseudomonas aeruginosa establishes chronic infection. Pseudomonas imbedded within biofilms is protected against the immune system, but maintains chronic inflammation that worsens disease symptoms. Aberrant NET release from recruited neutrophils was found in CF, but the underlying mechanisms remain unclear. One of the most important Pseudomonas virulence factors is pyocyanin, a redox-active pigment that has been associated with diminished lung function in CF. Here we show that pyocyanin promotes NET formation in a time- and dose-dependent manner. Most CF Pseudomonas clinical isolates tested produce pyocyanin in vitro. Pyocyanin-derived reactive oxygen species are required for its NET release. Inhibitor experiments demonstrated involvement of Jun N-terminal Kinase (JNK) and phosphatidylinositol 3-Kinase (PI3K) in pyocyanin-induced NET formation. Pyocyanin-induced NETs also require the NADPH oxidase because NET release in chronic granulomatous disease neutrophils was greatly reduced. Comparison of neutrophils from gp91phox- and p47phox-deficient patients revealed that pyocyanin-triggered NET formation is proportional to their residual superoxide production. Our studies identify pyocyanin as the first secreted bacterial toxin that enhances NET formation. The involvement of NADPH oxidase in pyocyanin-induced NET formation represents a novel mechanism of pyocyanin toxicity.

Rada, Balazs; Jendrysik, Meghan A.; Pang, Lan; Hayes, Craig P.; Yoo, Dae-goon; Park, Jonathan J.; Moskowitz, Samuel M.; Malech, Harry L.; Leto, Thomas L.

2013-01-01

265

NADPH oxidase inhibits the pathogenesis of systemic lupus erythematosus  

PubMed Central

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by loss of tolerance to self nucleic acids. The source of autoantigen that drives disease onset and progression is unclear. A candidate source of autoantigen is the Neutrophil Extracellular Trap (NET), which results in the release of nucleic acids into the extracellular environment, generating a structure composed of DNA coated with antimicrobial proteins. Based on in vitro and patient correlative studies, several groups have suggested NETs may provide lupus autoantigens. The observation that NET release (NETosis) relies on activity of the phagocyte NAPDH oxidase (Nox2) in neutrophils of both humans and mice provided a genetic strategy to test this hypothesis in vivo. To do so, we have crossed an X-linked nox2 null allele onto the lupus-prone MRL.Faslpr genetic background and assessed immune activation, autoantibody generation, and SLE pathology. Strikingly, and counter to the prevailing hypothesis, Nox2-deficient lupus-prone mice have markedly exacerbated lupus, including increased spleen weight, increased renal disease, and elevated and altered autoantibody profiles. Intriguingly, heterozygous female mice, which have Nox2-deficiency in 50% of neutrophils, also had exacerbated lupus and altered autoantibody patterns, suggesting that failure to undergo normal Nox2-dependent cell death may result in release of immunogenic self-constituents that stimulate lupus. Our results indicate that NETosis does not contribute to SLE in vivo, and rather that Nox2 acts to inhibit disease pathogenesis.

Campbell, A. M.; Kashgarian, M.; Shlomchik, M. J.

2013-01-01

266

Regional expression of NO synthase, NAD(P)H oxidase and superoxide dismutase in the rat brain.  

PubMed

Nitric oxide (NO) derived from the endothelial NO synthase (eNOS) contributes to regulation of cerebral circulation, whereas that produced by neuronal NOS (nNOS) participates in the regulation of brain function. In particular, NO plays an important role in modulation of sympathetic activity and hence central regulation of arterial pressure. Superoxide derived from NAD(P)H oxidase avidly reacts with and inactivates NO and, thereby, modulates its bioavailability. Calmodulin (CM) is required for activation of NOS and soluble guanylate cyclase (sGC) serves as a NO receptor. Superoxide is dismutated to H2O2 by superoxide dismutase (SOD) and H2O2 is converted to H2O by catalase or glutathione peroxidase (GPX). Given the importance of NO in the regulation of brain perfusion and function, we undertook the present study to determine the relative expressions of immunodetectable nNOS, eNOS, CM, sGC, NAD(P)H oxidase and SOD by Western analysis in different regions of the normal rat brain. nNOS was abundantly expressed in the pons cerebellum and hypothalamus and less so in the cortex and medulla. sGC abundance was highest in the hypothalamus and pons, and lowest in the cerebellum and medulla. eNOS and calmodulin were equally abundant in all regions. NAD(P)H oxide was most abundant in the pons compared to other regions. Cytoplasmic SOD was equally distributed among different regions but catalase and GPX were more abundant in pons, hypothalamus and medulla and less so in the cortex and cerebellum. Thus, the study documented regional distributions of NOS, NAD(P)H oxidase, antioxidant enzymes, sGC and calmodulin which collectively regulate production and biological activities of NO and superoxide, the two important small molecular size signaling molecules. PMID:17196179

Campese, Vito M; Sindhu, Ram K; Ye, Shaohua; Bai, Yongli; Vaziri, Nosratola D; Jabbari, Bahman

2006-12-28

267

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

268

Ethanol preconditioning protects against ischemia/reperfusion-induced brain damage: Role of NADPH oxidase-derived ROS  

PubMed Central

Ethanol preconditioning (EtOH-PC) refers to a phenomenon in which tissues are protected from the deleterious effects of ischemia/reperfusion (I/R) by prior ingestion of ethanol at low to moderate levels. In this study, we tested whether prior (24 hrs) administration of ethanol as a single bolus that produced a peak plasma concentration of 42-46 mg/dl in gerbils would offer protective effects on neuronal damage due to cerebral I/R. In addition, we also tested whether reactive oxygen species (ROS)-derived from NADPH oxidase played a role as an initiator of these putative protective effects. Groups of gerbils were administered either ethanol or the same volume of water by gavage 24 hrs prior to transient global cerebral ischemia induced by occlusion of both common carotid arteries (CCA) for 5 min. In some experiments, apocynin, a specific inhibitor of NADPH oxidase, was administered (5 mg/kg body wt, i.p.) 10 min before ethanol administration. EtOH-PC ameliorated behavioral deficit induced by cerebral I/R and protected the brain against I/R-induced delayed neuronal death (DND), neuronal and dendritic degeneration, oxidative DNA damage, and glial cell activation. These beneficial effects were attenuated by apocynin treatment coincident with ethanol administration. Ethanol ingestion was associated with translocation of the NADPH oxidase subunit, p67phox, from hippocampal cytosol fraction to membrane, increased NADPH oxidase activity in hippocampus within the first hour after gavage, and increased lipid peroxidation (4-hydroxy-2-nonenal, HNE) in plasma and hippocampus within the first 2 hrs after gavage. These effects were also inhibited by concomitant apocynin treatment. Our data are consistent with the hypothesis that antecedent ethanol ingestion at socially-relevant levels induces neuroprotective effects in I/R by a mechanism that is triggered by ROS produced through NADPH oxidase. Our results further suggest the possibility that preconditioning with other pharmacological agents that induce a mild oxidative stress may have similar therapeutic value for suppressing stroke-mediated damage in brain.

Wang, Qun; Sun, Albert Y.; Simonyi, Agnes; Kalogeris, Theodore J.; Miller, Dennis K.; Sun, Grace Y.; Korthuis, Ronald J.

2007-01-01

269

Annexin Peptide Ac2-26 Suppresses TNF?-Induced Inflammatory Responses via Inhibition of Rac1-Dependent NADPH Oxidase in Human Endothelial Cells  

PubMed Central

The anti-inflammatory peptide annexin-1 binds to formyl peptide receptors (FPR) but little is known about its mechanism of action in the vasculature. Here we investigate the effect of annexin peptide Ac2-26 on NADPH oxidase activity induced by tumour necrosis factor alpha (TNF?) in human endothelial cells. Superoxide release and intracellular reactive oxygen species (ROS) production from NADPH oxidase was measured with lucigenin-enhanced chemiluminescence and 2?,7?-dichlorodihydrofluorescein diacetate, respectively. Expression of NADPH oxidase subunits and intracellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) were determined by real-time PCR and Western blot analysis. Promoter activity of nuclear factor kappa B (NF?B) was measured by luciferase activity assay. TNF? stimulated NADPH-dependent superoxide release, total ROS formation and expression of ICAM-1and VCAM-1. Pre-treatment with N-terminal peptide of annexin-1 (Ac2-26, 0.5–1.5 µM) reduced all these effects, and the inhibition was blocked by the FPRL-1 antagonist WRW4. Furthermore, TNF?-induced NF?B promoter activity was attenuated by both Ac2-26 and NADPH oxidase inhibitor diphenyliodonium (DPI). Surprisingly, Nox4 gene expression was reduced by TNF? whilst expression of Nox2, p22phox and p67phox remained unchanged. Inhibition of NADPH oxidase activity by either dominant negative Rac1 (N17Rac1) or DPI significantly attenuated TNF?-induced ICAM-1and VCAM-1 expression. Ac2-26 failed to suppress further TNF?-induced expression of ICAM-1 and VCAM-1 in N17Rac1-transfected cells. Thus, Ac2-26 peptide inhibits TNF?-activated, Rac1-dependent NADPH oxidase derived ROS formation, attenuates NF?B pathways and ICAM-1 and VCAM-1 expression in endothelial cells. This suggests that Ac2-26 peptide blocks NADPH oxidase activity and has anti-inflammatory properties in the vasculature which contributes to modulate in reperfusion injury inflammation and vascular disease.

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

2013-01-01

270

Annexin peptide Ac2-26 suppresses TNF?-induced inflammatory responses via inhibition of Rac1-dependent NADPH oxidase in human endothelial cells.  

PubMed

The anti-inflammatory peptide annexin-1 binds to formyl peptide receptors (FPR) but little is known about its mechanism of action in the vasculature. Here we investigate the effect of annexin peptide Ac2-26 on NADPH oxidase activity induced by tumour necrosis factor alpha (TNF?) in human endothelial cells. Superoxide release and intracellular reactive oxygen species (ROS) production from NADPH oxidase was measured with lucigenin-enhanced chemiluminescence and 2',7'-dichlorodihydrofluorescein diacetate, respectively. Expression of NADPH oxidase subunits and intracellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) were determined by real-time PCR and Western blot analysis. Promoter activity of nuclear factor kappa B (NF?B) was measured by luciferase activity assay. TNF? stimulated NADPH-dependent superoxide release, total ROS formation and expression of ICAM-1and VCAM-1. Pre-treatment with N-terminal peptide of annexin-1 (Ac2-26, 0.5-1.5 µM) reduced all these effects, and the inhibition was blocked by the FPRL-1 antagonist WRW4. Furthermore, TNF?-induced NF?B promoter activity was attenuated by both Ac2-26 and NADPH oxidase inhibitor diphenyliodonium (DPI). Surprisingly, Nox4 gene expression was reduced by TNF? whilst expression of Nox2, p22phox and p67phox remained unchanged. Inhibition of NADPH oxidase activity by either dominant negative Rac1 (N17Rac1) or DPI significantly attenuated TNF?-induced ICAM-1and VCAM-1 expression. Ac2-26 failed to suppress further TNF?-induced expression of ICAM-1 and VCAM-1 in N17Rac1-transfected cells. Thus, Ac2-26 peptide inhibits TNF?-activated, Rac1-dependent NADPH oxidase derived ROS formation, attenuates NF?B pathways and ICAM-1 and VCAM-1 expression in endothelial cells. This suggests that Ac2-26 peptide blocks NADPH oxidase activity and has anti-inflammatory properties in the vasculature which contributes to modulate in reperfusion injury inflammation and vascular disease. PMID:23637767

Peshavariya, Hitesh M; Taylor, Caroline J; Goh, Celeste; Liu, Guei-Sheung; Jiang, Fan; Chan, Elsa C; Dusting, Gregory J

2013-04-24

271

Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes.  

PubMed Central

The NADPH oxidase of phagocytic cells is important for the efficient killing and digestion of ingested microbes. A very unusual low-potential cytochrome b (b-245) is the only redox molecule to have been identified in this system. The FAD-containing flavoprotein that binds NADPH and transfers electrons to the cytochrome has eluded identification for three decades. We show here that the haem/FAD ratio in the membranes does not change significantly on activation of this oxidase, indicating that the FAD is present in the membranes from the outset and not recruited from the cytosol. The FAD content of membranes from cells of patients with X-linked chronic granulomatous disease (CGD) lacking the cytochrome b was roughly one-quarter of that in normal subjects and in autosomal recessive CGD patients lacking the cytosolic protein p47-phox. Similar low amounts of FAD were present in uninduced promyelocytic (HL60) cells, suggesting that the low amount of FAD in cells from X-CGD patients was probably unrelated to this oxidase system. Cytochrome b-245 appears to bind both the haem and FAD, in a molar ratio of 2:1. The e.p.r. signal of the purified cytochrome was weak and had an asymmetric g(z) peak at g = 3.31. The purified cytochrome could be partially reflavinated (about 20%) in the presence of lipid. Amino acid sequence homology was detected between the beta-subunit of this cytochrome b and the ferredoxin-NADP+ reductase (FNR) family of reductases in the putative NADPH- and FAD-binding sites. 32P-labelled 2-azido-NADP was used as a photoaffinity label for the NADPH-binding site. Labelling that was competed off with NADP was observed in the region of the beta-subunit of the cytochrome. No labelling was seen in this region in X-CGD in three subjects in whom this cytochrome was missing and in a third in whom it was present but bore a Pro-His transposition in the putative NADPH-binding site. These studies indicate that cytochrome b-245 is a flavocytochrome, the first described in higher eukaryotic cells, bearing the complete electron-transporting apparatus of the NADPH oxidase. Images Fig. 5.

Segal, A W; West, I; Wientjes, F; Nugent, J H; Chavan, A J; Haley, B; Garcia, R C; Rosen, H; Scrace, G

1992-01-01

272

A Medicago truncatula NADPH oxidase is involved in symbiotic nodule functioning.  

PubMed

The plant plasma membrane-localized NADPH oxidases, known as respiratory burst oxidase homologues (RBOHs), appear to play crucial roles in plant growth and development. They are involved in important processes, such as root hair growth, plant defence reactions and abscisic acid signalling. Using sequence similarity searches, we identified seven putative RBOH-encoding genes in the Medicago truncatula genome. A phylogenetic reconstruction showed that Rboh gene duplications occurred in legume species. We analysed the expression of these MtRboh genes in different M. truncatula tissues: one of them, MtRbohA, was significantly up-regulated in Sinorhizobium meliloti-induced symbiotic nodules. MtRbohA expression appeared to be restricted to the nitrogen-fixing zone of the functional nodule. Moreover, using S. meliloti bacA and nifH mutants unable to form efficient nodules, a strong link between nodule nitrogen fixation and MtRbohA up-regulation was shown. MtRbohA expression was largely enhanced under hypoxic conditions. Specific RNA interference for MtRbohA provoked a decrease in the nodule nitrogen fixation activity and the modulation of genes encoding the microsymbiont nitrogenase. These results suggest that hypoxia, prevailing in the nodule-fixing zone, may drive the stimulation of MtRbohA expression, which would, in turn, lead to the regulation of nodule functioning. PMID:21155825

Marino, Daniel; Andrio, Emilie; Danchin, Etienne G J; Oger, Elodie; Gucciardo, Sébastien; Lambert, Annie; Puppo, Alain; Pauly, Nicolas

2010-12-13

273

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

PubMed

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. PMID:12958034

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

2003-09-04

274

NADPH oxidase modulates MHC class II antigen presentation by B cells  

PubMed Central

Summary Phagocyte NADPH oxidase plays a key role in pathogen clearance via reactive oxygen species (ROS) production. Defects in oxidase function result in chronic granulomatous disease (CGD) with hallmark recurrent microbial infections and inflammation. The oxidase?s role in the adaptive immune response is not well-understood. Class II presentation of cytoplasmic and exogenous Ag to CD4+ T cells was impaired in human B cells with reduced oxidase p40phox subunit expression. Naturally arising mutations which compromise p40phox function in a CGD patient also perturbed class II Ag presentation and intracellular ROS production. Reconstitution of patient B cells with wild-type, but not a mutant, p40phox allele restored exogenous Ag presentation and intracellular ROS generation. Remarkably, class II presentation of epitopes from membrane Ag was robust in p40phox-deficient B cells. These studies reveal a role for NADPH oxidase and p40phox in skewing epitope selection and T cell recognition of self Ag.

Crotzer, Victoria L.; Matute, Juan D.; Arias, Andres A.; Zhao, Heng; Quilliam, Lawrence A.; Dinauer, Mary C.; Blum, Janice S.

2012-01-01

275

NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action  

PubMed Central

BACKGROUND Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood. EXPERIMENTAL APPROACH Celastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins. KEY RESULTS Low concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration–response curves exhibiting higher Hill coefficients and lower IC50 values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC50 of 1.24 and 8.4 µM for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47phox translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47phox and disrupted the binding of the proline rich region of p22phox to the tandem SH3 domain of p47phox and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively. CONCLUSIONS AND IMPLICATIONS These results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome.

Jaquet, Vincent; Marcoux, Julien; Forest, Eric; Leidal, Kevin G; McCormick, Sally; Westermaier, Yvonne; Perozzo, Remo; Plastre, Olivier; Fioraso-Cartier, Laetitia; Diebold, Becky; Scapozza, Leonardo; Nauseef, William M; Fieschi, Franck; Krause, Karl-Heinz; Bedard, Karen

2011-01-01

276

Attenuating effect of angiotensin-(1-7) on angiotensin II-mediated NAD(P)H oxidase activation in type 2 diabetic nephropathy of KK-A(y)/Ta mice.  

PubMed

ANG-(1-7) is associated with vasodilation and nitric oxide synthase stimulation. However, the role of ANG-(1-7) in type 2 diabetes mellitus is unknown. In this study, we examined the hypothesis that ANG-(1-7) attenuates ANG II-induced reactive oxygen species stress (ROS)-mediated injury in type 2 diabetic nephropathy of KK-A(y)/Ta mice. KK-A(y)/Ta mice were divided into four groups: 1) a control group; 2) ANG II infusion group; 3) ANG II+ANG-(1-7) coinfusion group; and 4) ANG II+ANG-(1-7)+d-Ala(7)-ANG-(1-7) (A779) coinfusion group. In addition, primary mesangial cells were cultured and then stimulated with 25 mM glucose with or without ANG II, ANG-(1-7), and A779. The ANG II+ANG-(1-7) coinfusion group showed a lower urinary albumin/creatinine ratio increase than the ANG II group. ANG-(1-7) attenuated ANG II-mediated NAD(P)H oxidase activation and ROS production in diabetic glomeruli and mesangial cells. ANG II-induced NF-?B and MAPK signaling activation was also attenuated by ANG-(1-7) in the mesangial cells. These findings were related to improved mesangial expansion and to fibronectin and transforming growth factor-?1 production in response to ANG II and suggest that ANG-(1-7) may attenuate ANG II-stimulated ROS-mediated injury in type 2 diabetic nephropathy. The ACE2-ANG-(1-7)-Mas receptor axis should be investigated as a novel target for treatment of type 2 diabetic nephropathy. PMID:21367916

Moon, Ju-Young; Tanimoto, Mitsuo; Gohda, Tomohito; Hagiwara, Shinji; Yamazaki, Takahiko; Ohara, Ikko; Murakoshi, Maki; Aoki, Tatsuya; Ishikawa, Yuji; Lee, Sang-Ho; Jeong, Kyung-Hwan; Lee, Tae-Won; Ihm, Chun-Gyoo; Lim, Sung Jig; Tomino, Yasuhiko

2011-03-02

277

The NADPH oxidase inhibitor diphenyleneiodonium is also a potent inhibitor of cholinesterases and the internal Ca2+ pump  

PubMed Central

Background and purpose: Diphenyleneiodonium (DPI) is often used as an NADPH oxidase inhibitor, but is increasingly being found to have unrelated side effects. We investigated its effects on smooth muscle contractions and the related mechanisms. Experimental approach: We studied isometric contractions in smooth muscle strips from bovine trachea. Cholinesterase activity was measured using a spectrophotometric assay; internal Ca2+ pump activity was assessed by Ca2+ uptake into smooth muscle microsomes. Key results: Contractions to acetylcholine were markedly enhanced by DPI (10?4 M), whereas those to carbachol (CCh) were not, suggesting a possible inhibition of cholinesterase. DPI markedly suppressed contractions evoked by CCh, KCl and 5-HT, and also unmasked phasic activity in otherwise sustained responses. Direct biochemical assays confirmed that DPI was a potent inhibitor of acetylcholinesterase and butyrylcholinesterase (IC50?8 × 10?6 M and 6 × 10?7 M, respectively), following a readily reversible, mixed non-competitive type of inhibition. The inhibitory effects of DPI on CCh contractions were not mimicked by another NADPH oxidase inhibitor (apocynin), nor the Src inhibitors PP1 or PP2, ruling out an action through the NADPH oxidase signalling pathway. Several features of the DPI-mediated suppression of agonist-evoked responses (i.e. suppression of peak magnitudes and unmasking of phasic activity) are similar to those of cyclopiazonic acid, an inhibitor of the internal Ca2+ pump. Direct measurement of microsomal Ca2+ uptake revealed that DPI modestly inhibits the internal Ca2+ pump. Conclusions and implications: DPI inhibits cholinesterase activity and the internal Ca2+ pump in tracheal smooth muscle.

Tazzeo, T; Worek, F; Janssen, LJ

2009-01-01

278

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

2013-03-17

279

Expression of NAD(P)H Oxidase Subunits and Their Contribution to Cardiovascular Damage in Aldosterone/Salt-Induced Hypertensive Rat  

PubMed Central

NAD(P)H oxidase plays an important role in hypertension and its complication in aldosterone-salt rat. We questioned whether NAD(P)H oxidase subunit expression and activity are modulated by aldosterone and whether this is associated with target-organ damage. Rats were infused with aldosterone (0.75 µg/hr/day) for 6 weeks and were given 0.9% NaCl±losartan (30 mg/kg/day), spironolactone (200 mg/kg/day), and apocynin (1.5 mM/L). Aldosterone-salt hypertension was prevented completely by spironolactone and modestly by losartan and apocynin. Aldosterone increased aortic NAD(P)H oxidase activity by 34% and spironolactone and losartan inhibited the activity. Aortic expression of the subunits p47phox, gp91phox, and p22phox increased in aldosterone-infused rats by 5.5, 4.7, and 3.2-fold, respectively, which was decreased completely by spironolactone and partially by losartan and apocynin. Therefore, the increased expression of NAD(P)H oxidase may contribute to cardiovascular damage in aldosterone-salt hypertension through the increased expression of each subunit.

Park, Young Mee; Lim, Bong Hee; Touyz, Rhian M.

2008-01-01

280

MAPK Signaling Regulates Nitric Oxide and NADPH Oxidase-Dependent Oxidative Bursts in Nicotiana benthamiana[W][OA  

PubMed Central

Nitric oxide (NO) and reactive oxygen species (ROS) act as signals in innate immunity in plants. The radical burst is induced by INF1 elicitin, produced by the oomycete pathogen Phytophthora infestans. NO ASSOCIATED1 (NOA1) and NADPH oxidase participate in the radical burst. Here, we show that mitogen-activated protein kinase (MAPK) cascades MEK2-SIPK/NTF4 and MEK1-NTF6 participate in the regulation of the radical burst. NO generation was induced by conditional activation of SIPK/NTF4, but not by NTF6, in Nicotiana benthamiana leaves. INF1- and SIPK/NTF4-mediated NO bursts were compromised by the knockdown of NOA1. However, ROS generation was induced by either SIPK/NTF4 or NTF6. INF1- and MAPK-mediated ROS generation was eliminated by silencing Respiratory Burst Oxidase Homolog B (RBOHB), an inducible form of the NADPH oxidase. INF1-induced expression of RBOHB was compromised in SIPK/NTF4/NTF6-silenced leaves. These results indicated that INF1 regulates NOA1-mediated NO and RBOHB-dependent ROS generation through MAPK cascades. NOA1 silencing induced high susceptibility to Colletotrichum orbiculare but not to P. infestans; conversely, RBOHB silencing decreased resistance to P. infestans but not to C. orbiculare. These results indicate that the effects of the radical burst on the defense response appear to be diverse in plant–pathogen interactions.

Asai, Shuta; Ohta, Kohji; Yoshioka, Hirofumi

2008-01-01

281

Normobaric hyperoxia protects the blood brain barrier through inhibiting Nox2 containing NADPH oxidase in ischemic stroke  

PubMed Central

Normobaric hyperoxia (NBO) has been shown to be neuro- and vaso-protective during ischemic stroke. However, the underlying mechanisms remain to be fully elucidated. Activation of NADPH oxidase critically contributes to ischemic brain damage via increase in ROS production. We herein tested the hypothesis that NBO protects the blood-brain barrier (BBB) via inhibiting gp91phox (or called Nox2) containing NADPH oxidase in a mouse model of middle cerebral artery occlusion (MCAO). Wild-type C57/BL6 mice and gp91phoxknockout mice were given NBO (95% O2) or normoxia (21% O2) during 90-min MCAO, followed by 22.5 hrs of reperfusion. BBB damage was quantified by measuring Evans blue extravasation. The protein levels of matrix metalloproteinase-9 (MMP-9), tight junction protein occludin and gp91phox were assessed with western blot. Gel zymography was used to assess the gelatinolytic activity of MMP-9. In the wild type mice, cerebral ischemia and reperfusion led to remarkable Evans blue extravasation, significantly increased gp91phox and MMP-9 levels and decreased occludin levels in the ischemic brain tissue. In gp91phox knockout mice, the changes in Evans blue extravasation, MMP-9 and occludin were at much smaller magnitudes when compared to the wild type. Importantly, NBO treatment significantly reduced the changes in all measured parameters in wild type mice, while did not cause additional reductions in these changes when gp91phox was knocked out. These results indicate that activation of Nox2 containing NADPH oxidase is implicated in the induction of MMP-9, loss of occludin and BBB disruption in ischemic stroke, and inhibition of Nox2 may be an important mechanism underlying NBO-afforded BBB protection.

2011-01-01

282

Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction  

Microsoft Academic Search

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are known to be involved in angiotensin II-induced hypertension\\u000a and endothelial dysfunction. Several Nox isoforms are expressed in the vessel wall, among which Nox2 is especially abundant\\u000a in the endothelium. Endothelial Nox2 levels rise during hypertension but little is known about the cell-specific role of endothelial\\u000a Nox2 in vivo. To address this question,

Colin E. Murdoch; Sara P. Alom-Ruiz; Minshu Wang; Min Zhang; Simon Walker; Bin Yu; Alison Brewer; Ajay M. Shah

2011-01-01

283

Aldosterone mediates angiotensin II-induced interstitial cardiac fibrosis via a Nox2-containing NADPH oxidase  

Microsoft Academic Search

Angiotensin (ANG) II (AngII) and aldo- sterone contribute to the development of interstitial cardiac fibrosis. We investigated the potential role of a Nox2-containing NADPH oxidase in aldosterone-in- duced fibrosis and the involvement of this mechanism in AngII-induced effects. Nox2\\/ mice were com- pared with matched wild-type controls (WT). In WT mice, subcutaneous (s.c.) AngII (1.1 mg\\/kg\\/day for 2 wk) significantly

Sofian Johar; Alison C. Cave; Anilkumar Narayanapanicker; David J. Grieve; Ajay M. Shah

2006-01-01

284

Relative contributions of mitochondria and NADPH oxidase to deoxycorticosterone acetate-salt hypertension in mice  

Microsoft Academic Search

We assessed the relative contribution of the mitochondrial respiratory chain and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase to deoxycorticosterone acetate (DOCA)-salt hypertension in mice. The daily mean arterial pressure was monitored by radiotelemetry in DOCA-salt-treated mice given vehicle or the mitochondrial respiratory chain complex I inhibitor rotenone. This treatment produced remarkable attenuation of DOCA-salt hypertension. Similar results were obtained with

Aihua Zhang; Zhanjun Jia; Ningning Wang; Tyson J Tidwell; Tianxin Yang

2011-01-01

285

Isoforms and Functions of NAD(P)H Oxidase at the Macula Densa  

Microsoft Academic Search

Macula densa cells produce superoxide (O2 ) during tubuloglomerular feedback primarily via NAD(P)H oxidase (NOX). The purpose of the present study was to determine NOXs expressed by the macula densa and the role of each one in NaCl-induced O2 production. To identify which isoforms are expressed, we applied single-cell RT-PCR to macula densa cells isolated by laser capture microdissection and

Rui Zhang; Pamela Harding; Jeffrey L. Garvin; Ramiro Juncos; Ed Peterson; Luis A. Juncos; Ruisheng Liu

2010-01-01

286

Superoxide Mediates Sympathoexcitation in Heart Failure Roles of Angiotensin II and NAD(P)H Oxidase  

Microsoft Academic Search

Chronic heart failure (CHF) is often associated with excitation of the sympathetic nervous system. This event is thought to be a negative predictor of survival in CHF. Sympathoexcitation and central angiotensin II (Ang II) have been causally linked. Recent studies have shown that NAD(P)H oxidase-derived reactive oxidant species (ROS) are important mediators of Ang II signaling. In the present study,

Lie Gao; Wei Wang; Yu-Long Li; Harold D. Schultz; Dongmei Liu; Kurtis G. Cornish; Irving H. Zucker

287

Superoxide dismutase, catalase, glutathione peroxidase and NADPH oxidase in lead-induced hypertension  

Microsoft Academic Search

Superoxide dismutase, catalase, glutathione peroxidase and NADPH oxidase in lead-induced hypertension.BackgroundEarlier studies from this laboratory have revealed the presence of oxidative stress and its role in the pathogenesis of lead-induced hypertension (HTN). We have further shown evidence of increased hydroxyl radical (·OH) and superoxide production in lead-treated rats and cultured endothelial cells. This study was designed to determine whether oxidative

Nosratola D Vaziri; Ching-Yi Lin; Farbod Farmand; Ram K Sindhu

2003-01-01

288

Leptin regulates cardiomyocyte contractile function through endothelin-1 receptor-NADPH oxidase pathway.  

PubMed

Leptin, the obese gene product, plays an important role in the regulation of cardiac function. However, the mechanism behind leptin-induced cardiomyocyte contractile response is poorly understood. This study was designed to examine whether endothelin-1 receptor and NADPH oxidase play any role in leptin-induced cardiac contractile response. Isolated murine cardiomyocytes were exposed to leptin (5, 50, and 100 nmol/L) for 60 minutes in the absence or presence of the ETA receptor antagonist BQ123 (1 micromol/L), the ETB receptor antagonist BQ788 (1 micromol/L), or the NADPH oxidase inhibitor apocynin (100 micromol/L) before mechanical function was studied. Superoxide levels were measured by dihydroethidium fluorescent dye and the superoxide dismutase-inhibitable reduction of cytochrome c. NADPH oxidase subunit expression (p22phox, p47phox, p67phox, and gp91phox) was evaluated with Western blot. Leptin depressed peak shortening and maximal velocity of shortening/relengthening (+/-dL/dt), prolonged the duration of relengthening (TR90) without affecting the time-to-peak cell shortening. Consistent with the mechanical characteristics, myocytes treated with leptin displayed a reduced electrically stimulated rise in intracellular Ca2+ (change in fura-2 fluorescence intensity) associated with a prolonged intracellular Ca2+ decay rate. All of the abnormalities were significantly attenuated by apocynin, BQ123, or BQ788. Intracellular superoxide generation was enhanced after leptin treatment, which was partially blocked by apocynin, BQ123, or BQ788. Leptin had no effect on p22phox and gp91phox but upregulated protein expression of p67phox and p47phox, both of which were inhibited by apocynin, BQ123, or BQ788. These results suggest that leptin suppresses cardiac contractile function in ventricular myocytes through the endothelin-1 receptor and NADPH oxidase-mediated pathway. PMID:16380530

Dong, Feng; Zhang, Xiaochun; Ren, Jun

2005-12-27

289

Reduced expression of the NADPH oxidase NOX4 is a hallmark of adipocyte differentiation  

Microsoft Academic Search

Adipocyte differentiation is a complex process regulated among other factors by insulin and the production of reactive oxygen species (ROS). NOX4 is a ROS generating NADPH oxidase enzyme mediating insulin?s action in 3T3L1 adipocytes. In the present paper we show that NOX4 is expressed at high levels both in white and brown preadipocytes and that differentiation into adipocytes results in

Sarah Mouche; Sanae Ben Mkaddem; Wei Wang; Masa Katic; Yu-Hua Tseng; Stephanie Carnesecchi; Klaus Steger; Michelangelo Foti; Christoph A. Meier; Patrick Muzzin; C. Ronald Kahn; Eric Ogier-Denis; Ildiko Szanto

2007-01-01

290

Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases  

Microsoft Academic Search

Reactive oxygen species (ROS) are important signal transduction molecules in ligand-induced signaling, regulation of cell growth, differentiation, apoptosis and motility. Recently NADPH oxidases (Nox) homologous to Nox2 (gp91phox) of phagocyte cytochrome b558 have been identified, which are an enzymatic source for ROS generation in epithelial cells. This study was undertaken to delineate the requirements for ROS generation by Nox4. Nox4,

Kendra D. Martyn; Linda M. Frederick; Katharina von Loehneysen; Mary C. Dinauer; Ulla G. Knaus

2006-01-01

291

NADPH oxidase inhibition reduces tubular sodium transport and improves kidney oxygenation in diabetes  

PubMed Central

Sustained hyperglycemia is associated with increased oxidative stress resulting in decreased intrarenal oxygen tension (Po2) due to increased oxygen consumption (Qo2). Chronic blockade of the main superoxide radicals producing system, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, normalizes Qo2 by isolated proximal tubular cells (PTC) and reduces proteinuria in diabetes. The aim was to investigate the effects of acute NADPH oxidase inhibition on tubular Na+ transport and kidney Po2 in vivo. Glomerular filtration rate (GFR), renal blood flow (RBF), filtration fraction (FF), Na+ excretion, fractional Li+ excretion, and intrarenal Po2 was measured in control and streptozotocin-diabetic rats during baseline and after acute NADPH oxidase inhibition using apocynin. The effects on tubular transporters were investigated using freshly isolated PTC. GFR was increased in diabetics compared with controls (2.2 ± 0.3 vs. 1.4 ± 0.1 ml·min?1·kidney?1). RBF was similar in both groups, resulting in increased FF in diabetics. Po2 was reduced in cortex and medulla in diabetic kidneys compared with controls (34.4 ± 0.7 vs. 42.5 ± 1.2 mmHg and 15.7 ± 1.2 vs. 25.5 ± 2.3 mmHg, respectively). Na+ excretion was increased in diabetics compared with controls (24.0 ± 4.7 vs. 9.0 ± 2.0 ?m·min?1·kidney?1). In controls, all parameters were unaffected. However, apocynin increased Na+ excretion (+112%) and decreased fractional lithium reabsorption (?10%) in diabetics, resulting in improved cortical (+14%) and medullary (+28%) Po2. Qo2 was higher in PTC isolated from diabetic rats compared with control. Apocynin, dimethylamiloride, and ouabain reduced Qo2, but the effects of combining apocynin with either dimethylamiloride or ouabain were not additive. In conclusion, NADPH oxidase inhibition reduces tubular Na+ transport and improves intrarenal Po2 in diabetes.

Persson, Patrik; Hansell, Peter

2012-01-01

292

NADPH oxidase(s): new source(s) of reactive oxygen species in the vascular system?  

Microsoft Academic Search

Reactive oxygen species play an important role in a variety of (patho)physiological vascular processes. Recent publications have produced evidence of a role for putative non-phagocyte NADP oxidase(s) in the vascular production of reactive oxygen species. In the present review, we discuss the detection of the different components of NADP oxidase(s) in the vascular system, together with the putative role of

L Van Heerebeek; C. Meischl; W. Stooker; C. J. L. M. Meijer; H. W. M. Niessen; D. Roos

2002-01-01

293

Methamphetamine alters occludin expression via NADPH oxidase-induced oxidative insult and intact caveolae.  

PubMed

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. This 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

2012-02-01

294

Hypoxia induces Kv channel current inhibition by increased NADPH oxidase-derived reactive oxygen species.  

PubMed

There is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary circulation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they are derived, and what the downstream targets of ROS are. We recently showed that the NADPH oxidase homolog NOX4 is upregulated in hypoxia-induced pulmonary hypertension in mice and contributes to the vascular remodeling in pulmonary hypertension. We here tested the hypothesis that NOX4 regulates K(v) channels via an increased ROS formation after prolonged hypoxia. We showed that (1) NOX4 is upregulated in hypoxia-induced pulmonary hypertension in rats and isolated rat pulmonary arterial smooth muscle cells (PASMC) after 3days of hypoxia, and (2) that NOX4 is a major contributor to increased reactive oxygen species (ROS) after hypoxia. Our data indicate colocalization of K(v)1.5 and NOX4 in isolated PASMC. The NADPH oxidase inhibitor and ROS scavenger apocynin as well as NOX4 siRNA reversed the hypoxia-induced decrease in K(v) current density whereas the protein levels of the channels remain unaffected by siNOX4 treatment. Determination of cysteine oxidation revealed increased NOX4-mediated K(v)1.5 channel oxidation. We conclude that sustained hypoxia decreases K(v) channel currents by a direct effect of a NOX4-derived increase in ROS. PMID:22222468

Mittal, Manish; Gu, Xiang Q; Pak, Oleg; Pamenter, Matthew E; Haag, Daniela; Fuchs, D Beate; Schermuly, Ralph T; Ghofrani, H A; Brandes, Ralf P; Seeger, Werner; Grimminger, Friedrich; Haddad, Gabriel G; Weissmann, Norbert

2011-12-16

295

NADPH oxidases regulate septin-mediated cytoskeletal remodeling during plant infection by the rice blast fungus  

PubMed Central

The rice blast fungus Magnaporthe oryzae infects plants with a specialized cell called an appressorium, which uses turgor to drive a rigid penetration peg through the rice leaf cuticle. Here, we show that NADPH oxidases (Nox) are necessary for septin-mediated reorientation of the F-actin cytoskeleton to facilitate cuticle rupture and plant cell invasion. We report that the Nox2–NoxR complex spatially organizes a heteroligomeric septin ring at the appressorium pore, required for assembly of a toroidal F-actin network at the point of penetration peg emergence. Maintenance of the cortical F-actin network during plant infection independently requires Nox1, a second NADPH oxidase, which is necessary for penetration hypha elongation. Organization of F-actin in appressoria is disrupted by application of antioxidants, whereas latrunculin-mediated depolymerization of appressorial F-actin is competitively inhibited by reactive oxygen species, providing evidence that regulated synthesis of reactive oxygen species by fungal NADPH oxidases directly controls septin and F-actin dynamics.

Ryder, Lauren S.; Dagdas, Yasin F.; Mentlak, Thomas A.; Kershaw, Michael J.; Thornton, Christopher R.; Schuster, Martin; Chen, Jisheng; Wang, Zonghua; Talbot, Nicholas J.

2013-01-01

296

The NOX toolbox: validating the role of NADPH oxidases in physiology and disease.  

PubMed

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis. PMID:22648375

Altenhöfer, Sebastian; Kleikers, Pamela W M; Radermacher, Kim A; Scheurer, Peter; Rob Hermans, J J; Schiffers, Paul; Ho, Heidi; Wingler, Kirstin; Schmidt, Harald H H W

2012-05-31

297

Relative contributions of mitochondria and NADPH oxidase to deoxycorticosterone acetate-salt hypertension in mice.  

PubMed

We assessed the relative contribution of the mitochondrial respiratory chain and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase to deoxycorticosterone acetate (DOCA)-salt hypertension in mice. The daily mean arterial pressure was monitored by radiotelemetry in DOCA-salt-treated mice given vehicle or the mitochondrial respiratory chain complex I inhibitor rotenone. This treatment produced remarkable attenuation of DOCA-salt hypertension. Similar results were obtained with other inhibitors of mitochondrial function, including 5-hydroxydecanoate (specific for mitochondrial potassium-ATP channels), benzylguanidine (complexes I and III), and the cell-permeable manganese tetrakis (4-benzoic acid) porphyrin (a mimic of mitochondrial superoxide dismutase). In parallel with the blood pressure-lowering effect of rotenone, the DOCA-salt-induced increases in urinary 8-isoprostane excretion and in reactive oxygen species production of isolated kidney mitochondria were both significantly attenuated. Conversely, the DOCA-salt-induced reduction of urinary nitrate/nitrite excretion was significantly elevated. Following DOCA-salt treatment, mice deficient in NADPH oxidase subunits gp91(phox) or p47(phox) exhibited a partial attenuation of the hypertensive response at early but not later time points. Thus, the mitochondrial respiratory chain is a major source of oxidative stress in DOCA-salt hypertension, whereas NADPH oxidase may have a relatively minor role during the early stage of hypertension. PMID:21368743

Zhang, Aihua; Jia, Zhanjun; Wang, Ningning; Tidwell, Tyson J; Yang, Tianxin

2011-03-02

298

Oxidative stress in atrial fibrillation: an emerging role of NADPH oxidase.  

PubMed

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

2013-05-02

299

Decreased neointimal formation in Nox2-deficient mice reveals a direct role for NADPH oxidase in the response to arterial injury  

Microsoft Academic Search

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced, in part, from NADPH oxidase in response to host invasion and tissue injury. Defects in NADPH oxidase impair host defense; however, the role of ROS and RNS in the response to tissue injury is not known. We addressed this issue by subjecting leukocyte oxidase (Nox2)-deficient (Nox2-\\/-) mice to arterial

Zhiping Chen; John F. Keaney Jr.; Eberhard Schulz; Bruce Levison; Lian Shan; Masashi Sakuma; Xiaobin Zhang; Can Shi; Stanley L. Hazen; Daniel I. Simon

2004-01-01

300

NADPH Oxidase-Dependent Reactive Oxygen Species Mediate Amplified TLR4 Signaling and Sepsis-Induced Mortality in Nrf2-deficient Mice  

PubMed Central

Sepsis syndrome is characterized by a dysregulated inflammatory response to infection. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. We previously showed that disruption of Nrf2, a master regulator of antioxidant defenses, caused a dysregulation of innate immune response that resulted in greater mortality in a polymicrobial sepsis and lipopolysaccharide (LPS) shock model; however, the underlying mechanisms are unclear. In the present study, compared to wild-type (Nrf2+/+) macrophages, we observed greater PKC-induced NADPH oxidase-dependent ROS generation in Nrf2-disrupted (Nrf2?/?) macrophages that was modulated by glutathione (GSH) levels. To address the NADPH oxidase-mediated hyper-inflammatory response and sepsis-induced lung injury and mortality in Nrf2?/? mice, we used double knockout mice lacking Nrf2 and NADPH oxidase subunit, gp91phox (Nrf2?/?//Gp91phox?/?). Compared to Nrf2+/+ macrophages, LPS induced greater activation of TLR4 as evident by TLR4 surface trafficking and downstream recruitment of MYD88 and TRIF in Nrf2?/? macrophages that was diminished by ablation of gp91phox. Similarly, phosphorylation of I?B and IRF3 as well as cytokine expression was markedly higher in Nrf2?/? macrophages, while it was similar in Nrf2+/+ and Nrf2?/?//Gp91phox?/?. In vivo studies showed greater LPS-induced pulmonary inflammation in Nrf2?/? mice that was significantly reduced by ablation of gp91phox. Furthermore, LPS shock and polymicrobial sepsis induced early and greater mortality in Nrf2?/? mice, while Nrf2?/?//Gp91phox?/? showed prolong survival. Together, these results demonstrate that Nrf2 is essential for the regulation of NADPH oxidase-dependent ROS-mediated TLR4 activation and lethal innate immune response in sepsis.

Kong, Xiaoni; Thimmulappa, Rajesh; Kombairaju, Ponvijay; Biswal, Shyam

2010-01-01

301

AT1 blockade prevents glucose-induced cardiac dysfunction in ventricular myocytes: role of the AT1 receptor and NADPH oxidase.  

PubMed

Enhanced tissue angiotensin (Ang) II levels have been reported in diabetes and might lead to cardiac dysfunction through oxidative stress. This study examined the effect of blocking the Ang II type 1 (AT1) receptor on high glucose-induced cardiac contractile dysfunction. Rat ventricular myocytes were maintained in normal- (NG, 5.5 mmol/L) or high- (HG, 25.5 mmol/L) glucose medium for 24 hours. Mechanical and intracellular Ca2+ properties were assessed as peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening (+/-dL/dt), and intracellular Ca2+ decay (tau). HG myocytes exhibited normal PS; decreased +/-dL/dt; and prolonged TPS, TR90, and tau. Interestingly, the HG-induced abnormalities were prevented with the AT1 blocker L-158,809 (10 to 1000 nmol/L) but not the Janus kinase-2 (JAK2) inhibitor AG-490 (10 to 100 micromol/L). The only effect of AT1 blockade on NG myocytes was enhanced PS at 1000 nmol/L. AT1 antagonist-elicited cardiac protection against HG was nullified by the NADPH oxidase activator sodium dodecyl sulfate (80 micromol/L) and mimicked by the NADPH oxidase inhibitors diphenyleneiodonium (10 micromol/L) or apocynin (100 micromol/L). Western blot analysis confirmed that the protein abundance of NADPH oxidase subunit p47phox and the AT1 but not the AT2 receptor was enhanced in HG myocytes. In addition, the HG-induced increase of p47phox was prevented by L-158,809. Enhanced reactive oxygen species production observed in HG myocytes was prevented by AT1 blockade or NADPH oxidase inhibition. Collectively, our data suggest that local Ang II, acting via AT1 receptor-mediated NADPH oxidase activation, is involved in hyperglycemia-induced cardiomyocyte dysfunction, which might play a role in diabetic cardiomyopathy. PMID:12847113

Privratsky, Jamie R; Wold, Loren E; Sowers, James R; Quinn, Mark T; Ren, Jun

2003-07-07

302

Insulin increases surface expression of TRPC6 channels in podocytes: role of NADPH oxidases and reactive oxygen species  

PubMed Central

Insulin receptors in podocytes are essential for normal kidney function. Here, we show that insulin evokes a rapid increase in the surface expression of canonical transient receptor potential-6 channel (TRPC6) channels in cultured podocytes, but caused a decrease in surface expression of TRPC5. These effects are accompanied by a marked increase in outwardly rectifying cationic currents that can be blocked by 10 ?M SKF96365 or 100 ?M La3+. Application of oleoyl-2-acetyl-sn-glycerol (OAG) also increased SKF96365- and La3+-sensitive cationic currents in podocytes. Importantly, current responses to a combination of OAG and insulin were the same amplitude as those evoked by either agent applied alone. This occlusion effect suggests that OAG and insulin are targeting the same population of channels. In addition, shRNA knockdown of TRPC6 markedly reduced cationic currents stimulated by insulin. The effects of insulin on TRPC6 were mimicked by treating podocytes with H2O2. Insulin treatment rapidly increased the generation of H2O2 in podocytes, and it increased the surface expression of the NADPH oxidase NOX4 in cultured podocytes. Basal and insulin-stimulated surface expression of TRPC6 were reduced by pretreatment with diphenylene iodonium, an inhibitor of NADPH oxidases and other flavin-dependent enzymes, by siRNA knockdown of NOX4, and by manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, a membrane-permeable mimetic of superoxide dismutase and catalase. These observations suggest that insulin increases generation of ROS in part through activation of NADPH oxidases, and that this step contributes to modulation of podocyte TRPC6 channels.

Kim, Eun Young; Anderson, Marc

2012-01-01

303

Low-Dose Dextromethorphan, a NADPH Oxidase Inhibitor, Reduces Blood Pressure and Enhances Vascular Protection in Experimental Hypertension  

PubMed Central

Background Vascular oxidative stress may be increased with age and aggravate endothelial dysfunction and vascular injury in hypertension. This study aimed to investigate the effects of dextromethorphan (DM), a NADPH oxidase inhibitor, either alone or in combination treatment, on blood pressure (BP) and vascular protection in aged spontaneous hypertensive rats (SHRs). Methodology/Principal Findings Eighteen-week-old WKY rats and SHRs were housed for 2 weeks. SHRs were randomly assigned to one of the 12 groups: untreated; DM monotherapy with 1, 5 or 25 mg/kg/day; amlodipine (AM, a calcium channel blocker) monotherapy with 1 or 5 mg/kg/day; and combination therapy of DM 1, 5 or 25 mg/kg/day with AM 1 or 5 mg/kg/day individually for 4 weeks. The in vitro effects of DM were also examined. In SHRs, AM monotherapy dose-dependently reduced arterial systolic BP. DM in various doses significantly and similarly reduced arterial systolic BP. Combination of DM with AM gave additive effects on BP reduction. DM, either alone or in combination with AM, improved aortic endothelial function indicated by ex vivo acetylcholine-induced relaxation. The combination of low-dose DM with AM gave most significant inhibition on aortic wall thickness in SHRs. Plasma total antioxidant status was significantly increased by all the therapies except for the combination of high-dose DM with high-dose AM. Serum nitrite and nitrate level was significantly reduced by AM but not by DM or the combination of DM with AM. Furthermore, in vitro treatment with DM reduced angiotensin II-induced reactive oxygen species and NADPH oxidase activation in human aortic endothelial cells. Conclusions/Significance Treatment of DM reduced BP and enhanced vascular protection probably by inhibiting vascular NADPH oxidase in aged hypertensive animals with or without AM treatment. It provides the potential rationale to a novel combination treatment with low-dose DM and AM in clinical hypertension.

Wu, Tao-Cheng; Chao, Chih-Yu; Lin, Shing-Jong; Chen, Jaw-Wen

2012-01-01

304

JNK and NADPH Oxidase Involved in Fluoride-Induced Oxidative Stress in BV-2 Microglia Cells  

PubMed Central

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 O2·? 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.

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

2013-01-01

305

Optimization of important early ADME(T) parameters of NADPH oxidase-4 inhibitor molecules.  

PubMed

Through their reactive oxygen species (ROS) producing function, NADPH oxidase (NOX) enzymes have been linked to several oxidative stress related diseases. In our recently published paper [1] we have already shown the NOX4 inhibitory effect of diverse, molecule sub-libraries and their biological importance. We also presented our work connected to potential anti-tumour molecules and the relationship between their biological activity and physico-chemical properties [2]. As an extension of these studies further physico-chemical and biological investigation has been carried out on a molecule group included NOX4 inhibitory chromanone compounds. Here we describe the optimization of early ADME(T) parameters determining lipophilicity, phospholipophilicity and permeability linked to structure-activity relationship. We prove that optimal lipo- and phospholipophilicty can be also determined in case of NOX4 inhibitors and a comparison will be made between the chemically similar isochromanone and chromanone molecular libraries. It will be also shown how to predict the effect of different substituents on permeability, lipo- and phospholipophilicity and also the biological differences between anti-tumour molecules and NOX4 inhibitors according to their penetration ability. PMID:22385185

Borbély, Gábor; Huszár, Ménika; Varga, Attila; Futosi, Krisztina; Mócsai, Attila; Orfi, László; Idei, Miklós; Mandl, József; Kéri, György; Vántus, Tibor

2012-03-01

306

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

2012-12-20

307

Urotensin II Inhibits Skeletal Muscle Glucose Transport Signaling Pathways via the NADPH Oxidase Pathway  

PubMed Central

Our previous studies have demonstrated that the urotensin (UII) and its receptor are up-regulated in the skeletal muscle of mice with type II diabetes mellitus (T2DM), but the significance of UII in skeletal muscle insulin resistance remains unknown. The purpose of this study was to investigate the effect of UII on NADPH oxidase and glucose transport signaling pathways in the skeletal muscle of mice with T2DM and in C2C12 mouse myotube cells. KK/upj-AY/J mice (KK) mice were divided into the following groups: KK group, with saline treatment for 2 weeks; KK+ urantide group, with daily 30 µg/kg body weight injections over the same time period of urantide, a potent urotensin II antagonist peptide; Non-diabetic C57BL/6J mice were used as normal controls. After urantide treatment, mice were subjected to an intraperitoneal glucose tolerance test, in addition to measurements of the levels of ROS, NADPH oxidase and the phosphorylated AKT, PKC and ERK. C2C12 cells were incubated with serum-free DMEM for 24 hours before conducting the experiments, and then administrated with 100 nM UII for 2 hours or 24 hours. Urantide treatment improved glucose tolerance, decreased the translocation of the NADPH subunits p40-phox and p47-phox, and increased levels of the phosphorylated PKC, AKT and ERK. In contrast, UII treatment increased ROS production and p47-phox and p67-phox translocation, and decreased the phosphorylated AKT, ERK1/2 and p38MAPK; Apocynin abrogated this effect. In conclusion, UII increased ROS production by NADPH oxidase, leading to the inhibition of signaling pathways involving glucose transport, such as AKT/PKC/ERK. Our data imply a role for UII at the molecular level in glucose homeostasis, and possibly in skeletal muscle insulin resistance in T2DM.

Wang, Hong-Xia; Wu, Xin-Rui; Yang, Hui; Yin, Chun-Lin; Shi, Li-Jin; Wang, Xue-Jiang

2013-01-01

308

Urotensin II Inhibits Skeletal Muscle Glucose Transport Signaling Pathways via the NADPH Oxidase Pathway.  

PubMed

Our previous studies have demonstrated that the urotensin (UII) and its receptor are up-regulated in the skeletal muscle of mice with type II diabetes mellitus (T2DM), but the significance of UII in skeletal muscle insulin resistance remains unknown. The purpose of this study was to investigate the effect of UII on NADPH oxidase and glucose transport signaling pathways in the skeletal muscle of mice with T2DM and in C2C12 mouse myotube cells. KK/upj-AY/J mice (KK) mice were divided into the following groups: KK group, with saline treatment for 2 weeks; KK+ urantide group, with daily 30 µg/kg body weight injections over the same time period of urantide, a potent urotensin II antagonist peptide; Non-diabetic C57BL/6J mice were used as normal controls. After urantide treatment, mice were subjected to an intraperitoneal glucose tolerance test, in addition to measurements of the levels of ROS, NADPH oxidase and the phosphorylated AKT, PKC and ERK. C2C12 cells were incubated with serum-free DMEM for 24 hours before conducting the experiments, and then administrated with 100 nM UII for 2 hours or 24 hours. Urantide treatment improved glucose tolerance, decreased the translocation of the NADPH subunits p40-phox and p47-phox, and increased levels of the phosphorylated PKC, AKT and ERK. In contrast, UII treatment increased ROS production and p47-phox and p67-phox translocation, and decreased the phosphorylated AKT, ERK1/2 and p38MAPK; Apocynin abrogated this effect. In conclusion, UII increased ROS production by NADPH oxidase, leading to the inhibition of signaling pathways involving glucose transport, such as AKT/PKC/ERK. Our data imply a role for UII at the molecular level in glucose homeostasis, and possibly in skeletal muscle insulin resistance in T2DM. PMID:24116164

Wang, Hong-Xia; Wu, Xin-Rui; Yang, Hui; Yin, Chun-Lin; Shi, Li-Jin; Wang, Xue-Jiang

2013-10-08

309

Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes  

Microsoft Academic Search

Stimulation of bovine polymorphonuclear leukocytes (PMN) with serum-opsonized zymosan (sOZ) induced the activation of p38 mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3-K) and sOZ-induced O2? production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PKC and wortmannin for PI3-K). They caused significant attenuation of sOZ-induced phosphorylation of p47phox as well. Flow

Tohru Yamamori; Osamu Inanami; Hajime Nagahata; Yu-Dong Cui; Mikinori Kuwabara

2000-01-01

310

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

Microsoft Academic Search

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

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

2010-01-01

311

NADPH oxidase elevations in pyramidal neurons drive psychosocial stress-induced neuropathology  

PubMed Central

Oxidative stress is thought to be involved in the development of behavioral and histopathological alterations in animal models of psychosis. Here we investigate the causal contribution of reactive oxygen species generation by the phagocyte NADPH oxidase NOX2 to neuropathological alterations in a rat model of chronic psychosocial stress. In rats exposed to social isolation, the earliest neuropathological alterations were signs of oxidative stress and appearance of NOX2. Alterations in behavior, increase in glutamate levels and loss of parvalbumin were detectable after 4 weeks of social isolation. The expression of the NOX2 subunit p47phox was markedly increased in pyramidal neurons of isolated rats, but below detection threshold in GABAergic neurons, astrocytes and microglia. Rats with a loss of function mutation in the NOX2 subunit p47phox were protected from behavioral and neuropathological alterations induced by social isolation. To test reversibility, we applied the antioxidant/NOX inhibitor apocynin after initiation of social isolation for a time period of 3 weeks. Apocynin reversed behavioral alterations fully when applied after 4 weeks of social isolation, but only partially after 7 weeks. Our results demonstrate that social isolation induces rapid elevations of the NOX2 complex in the brain. Expression of the enzyme complex was strongest in pyramidal neurons and a loss of function mutation prevented neuropathology induced by social isolation. Finally, at least at early stages, pharmacological targeting of NOX2 activity might reverse behavioral alterations.

Schiavone, S; Jaquet, V; Sorce, S; Dubois-Dauphin, M; Hultqvist, M; Backdahl, L; Holmdahl, R; Colaianna, M; Cuomo, V; Trabace, L; Krause, K-H

2012-01-01

312

Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice.  

PubMed

Hemangiomas are the most common type of tumor in infants. As they are endothelial cell-derived neoplasias, their growth can be regulated by the autocrine-acting Tie2 ligand angiopoietin 2 (Ang2). Using an experimental model of human hemangiomas, in which polyoma middle T-transformed brain endothelial (bEnd) cells are grafted subcutaneously into nude mice, we compared hemangioma growth originating from bEnd cells derived from wild-type, Ang2+/-, and Ang2-/- mice. Surprisingly, Ang2-deficient bEnd cells formed endothelial tumors that grew rapidly and were devoid of the typical cavernous architecture of slow-growing Ang2-expressing hemangiomas, while Ang2+/- cells were greatly impaired in their in vivo growth. Gene array analysis identified a strong downregulation of NADPH oxidase 4 (Nox4) in Ang2+/- cells. Correspondingly, lentiviral silencing of Nox4 in an Ang2-sufficient bEnd cell line decreased Ang2 mRNA levels and greatly impaired hemangioma growth in vivo. Using a structure-based approach, we identified fulvenes as what we believe to be a novel class of Nox inhibitors. We therefore produced and began the initial characterization of fulvenes as potential Nox inhibitors, finding that fulvene-5 efficiently inhibited Nox activity in vitro and potently inhibited hemangioma growth in vivo. In conclusion, the present study establishes Nox4 as a critical regulator of hemangioma growth and identifies fulvenes as a potential class of candidate inhibitor to therapeutically interfere with Nox function. PMID:19620773

Bhandarkar, Sulochana S; Jaconi, Marisa; Fried, Levi E; Bonner, Michael Y; Lefkove, Benjamin; Govindarajan, Baskaran; Perry, Betsy N; Parhar, Ravi; Mackelfresh, Jamie; Sohn, Allie; Stouffs, Michael; Knaus, Ulla; Yancopoulos, George; Reiss, Yvonne; Benest, Andrew V; Augustin, Hellmut G; Arbiser, Jack L

2009-07-13

313

Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice  

PubMed Central

Hemangiomas are the most common type of tumor in infants. As they are endothelial cell–derived neoplasias, their growth can be regulated by the autocrine-acting Tie2 ligand angiopoietin 2 (Ang2). Using an experimental model of human hemangiomas, in which polyoma middle T–transformed brain endothelial (bEnd) cells are grafted subcutaneously into nude mice, we compared hemangioma growth originating from bEnd cells derived from wild-type, Ang2+/–, and Ang2–/– mice. Surprisingly, Ang2-deficient bEnd cells formed endothelial tumors that grew rapidly and were devoid of the typical cavernous architecture of slow-growing Ang2-expressing hemangiomas, while Ang2+/– cells were greatly impaired in their in vivo growth. Gene array analysis identified a strong downregulation of NADPH oxidase 4 (Nox4) in Ang2+/– cells. Correspondingly, lentiviral silencing of Nox4 in an Ang2-sufficient bEnd cell line decreased Ang2 mRNA levels and greatly impaired hemangioma growth in vivo. Using a structure-based approach, we identified fulvenes as what we believe to be a novel class of Nox inhibitors. We therefore produced and began the initial characterization of fulvenes as potential Nox inhibitors, finding that fulvene-5 efficiently inhibited Nox activity in vitro and potently inhibited hemangioma growth in vivo. In conclusion, the present study establishes Nox4 as a critical regulator of hemangioma growth and identifies fulvenes as a potential class of candidate inhibitor to therapeutically interfere with Nox function.

Bhandarkar, Sulochana S.; Jaconi, Marisa; Fried, Levi E.; Bonner, Michael Y.; Lefkove, Benjamin; Govindarajan, Baskaran; Perry, Betsy N.; Parhar, Ravi; Mackelfresh, Jamie; Sohn, Allie; Stouffs, Michael; Knaus, Ulla; Yancopoulos, George; Reiss, Yvonne; Benest, Andrew V.; Augustin, Hellmut G.; Arbiser, Jack L.

2009-01-01

314

Implication of NADPH Oxidases in the Early Inflammation Process Generated by Cystic Fibrosis Cells  

PubMed Central

In cystic fibrosis (CF) patients, pulmonary inflammation is a major cause of morbidity and mortality. The aim of this study was to further investigate whether oxidative stress could be involved in the early inflammatory process associated with CF pathogenesis. We used a model of CFTR defective epithelial cell line (IB3-1) and its reconstituted CFTR control (S9) cell line cultured in various ionic conditions. This study showed that IB3-1 and S9 cells expressed the NADPH oxidases (NOXs) DUOX1/2 and NOX2 at the same level. Nevertheless, several parameters participating in oxidative stress (increased ROS production and apoptosis, decreased total thiol content) were observed in IB3-1 cells cultured in hypertonic environment as compared to S9 cells and were inhibited by diphenyleneiodonium (DPI), a well-known inhibitor of NOXs; besides, increased production of the proinflammatory cytokines IL-6 and IL-8 by IB3-1 cells was also inhibited by DPI as compared to S9 cells. Furthermore, calcium ionophore (A23187), which upregulates DUOX and NOX2 activities, strongly induced oxidative stress and IL-8 and IL-6 overexpression in IB3-1 cells. All these events were suppressed by DPI, supporting the involvement of NOXs in the oxidative stress, which can upregulate proinflammatory cytokine production by the airway CFTR-deficient cells and trigger early pulmonary inflammation in CF patients.

Pongnimitprasert, Nushjira; Hurtado, Margarita; Lamari, Foudil; El Benna, Jamel; Dupuy, Corinne; Fay, Michele; Foglietti, Marie-Jose; Bernard, Maguy; Gougerot-Pocidalo, Marie-Anne; Braut-Boucher, Francoise

2012-01-01

315

C-Reactive Protein Inhibits Endothelium-Dependent NO-Mediated Dilation in Coronary Arterioles by Activating p38 Kinase and NAD(P)H Oxidase  

Microsoft Academic Search

Objective—Elevated levels of C-reactive protein (CRP), a proinflammatory marker, are associated with reduced systemic endothelium-dependent NO-mediated dilation in patients with coronary artery disease; however, the direct effect of CRP on coronary microvascular reactivity remains unknown. Herein, we examined whether CRP can modulate endotheli- um-dependent NO-mediated dilation of coronary arterioles and whether proinflammatory signaling pathways such as stress-activated protein kinases (p38

Erion Qamirani; Yi Ren; Lih Kuo; Travis W. Hein

2010-01-01

316

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

317

The Role of NADPH Oxidase 1-Derived Reactive Oxygen Species in Paraquat-Mediated Dopaminergic Cell Death  

PubMed Central

Abstract Oxidative stress is the common downstream effect of a variety of environmental neurotoxins that are strongly implicated in the pathogenesis of Parkinson's disease. We demonstrate here that the activation of NADPH oxidase 1 (Nox1), a specialized superoxide-generating enzyme complex, plays a key role in the oxidative stress and subsequent dopaminergic cell death elicited by paraquat. Paraquat increased the expression of Nox1 in a concentration-dependent manner in rat dopaminergic N27 cells. Rac1, a key component necessary for Nox1-mediated superoxide generation, also was activated by paraquat. Paraquat-induced reactive oxygen species generation and dopaminergic cell death were significantly reduced after pretreatment with apocynin, a putative NADPH oxidase inhibitor, and Nox1 knockdown with siRNA. Male C57BL/6 mice received intraperitoneal (IP) injections of paraquat (10?mg/kg) once every 3 days and showed increased Nox1 levels in the substantia nigra as well as a 35% reduction in tyrosine hydroxylase–positive dopaminergic neurons 5 days after the last injection. Preadministration of apocynin (200?mg/kg, IP) led to a significant decrease in dopaminergic neuronal loss. Our results suggest that Nox1-generated superoxide is implicated in the oxidative stress elicited by paraquat in DA cells, and it can serve as a novel target for pharmacologic intervention. Antioxid. Redox Signal. 11, 2105–2118.

Cristovao, Ana Clara; Choi, Dong-Hee; Baltazar, Graca; Beal, M. Flint

2009-01-01

318

7-Dehydrocholesterol Enhances Ultraviolet A-Induced Oxidative Stress in Keratinocytes: Roles of Nadph Oxidase, Mitochondria and Lipid Rafts  

PubMed Central

Long wavelength solar UVA radiation stimulates formation of reactive oxygen species (ROS) and prostaglandin E2 (PGE2), which are involved in skin photosensitivity and tumor promotion. High levels of 7-dehydrocholesterol (7-DHC), the precursor to cholesterol, cause exaggerated photosensitivity to UVA in patients with Smith-Lemli-Opitz syndrome (SLOS). Partially replacing cholesterol with 7-DHC in keratinocytes rapidly (<5 min) increased UVA-induced ROS, intracellular calcium, phospholipase A2 activity, PGE2, and NADPH oxidase activity. UVA-induced ROS and PGE2 production were inhibited in these cells by depleting the Nox1 subunit of NADPH oxidase using siRNA or using a mitochondrial radical quencher, MitoQ. Partial replacement of cholesterol with 7-DHC also disrupted membrane lipid raft domains, although depletion of cholesterol, which also disrupts lipid rafts, did not affect UVA-induced increases in ROS and PGE2. Phospholipid liposomes containing 7-DHC were more rapidly oxidized by a free radical mechanism than those containing cholesterol. These results indicate that 7-DHC enhances rapid UVA-induced ROS and PGE2 formation by enhancing free radical-mediated membrane lipid oxidation and suggests that this mechanism might underlie the UVA-photosensitivity in SLOS.

Valencia, Antonio; Rajadurai, Anpuchchelvi; Carle, A. Bjorn; Kochevar, Irene E.

2006-01-01

319

NADPH Oxidase 4 Mediates Insulin-Stimulated HIF-1? and VEGF Expression, and Angiogenesis In Vitro  

PubMed Central

Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 diabetes patients and is related to VEGF expression. Reactive oxygen species (ROS) have been shown to be involved in HIF-1? and VEGF expression induced by insulin, but the role of specific ROS sources has not been fully elucidated. In this study we examined the role of NADPH oxidase subunit 4 (Nox4) in insulin-stimulated HIF-1? and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs). Here we demonstrate that knockdown of Nox4 by siRNA reduced insulin-stimulated ROS generation, the tyrosine phosphorylation of IR-? and IRS-1, but did not change the serine phosphorylation of IRS-1. Nox4 gene silencing had a much greater inhibitory effect on insulin-induced AKT activation than ERK1/2 activation, whereas it had little effect on the expression of the phosphatases such as MKP-1 and SHIP. Inhibition of Nox4 expression inhibited the transcriptional activity of VEGF through HIF-1. Overexpression of wild-type Nox4 was sufficient to increase VEGF transcriptional activity, and further enhanced insulin-stimulated the activation of VEGF. Downregulation of Nox4 expression decreased insulin-stimulated mRNA and protein expression of HIF-1?, but did not change the rate of HIF-1? degradation. Inhibition of Nox4 impaired insulin-stimulated VEGF expression, cell migration, cell proliferation, and tube formation in HMVECs. Our data indicate that Nox4-derived ROS are essential for HIF-1?-dependent VEGF expression, and angiogenesis in vitro induced by insulin. Nox4 may be an attractive therapeutic target for diabetic retinopathy caused by intensive insulin treatment.

Meng, Dan; Mei, Aihong; Liu, Junxu; Kang, Xueling; Shi, Xianglin; Qian, Ruizhe; Chen, Sifeng

2012-01-01

320

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

PubMed

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; Machado, Moara; Magalhăes, 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

2013-07-02

321

Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4  

PubMed Central

Physiological sensing of O2 tension (partial O2 pressure, pO2) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO2 to altered calcium release through the ryanodine receptor–Ca2+-release channel (RyR1). Reactive oxygen species are generated in proportion to pO2 by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca2+ release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle. Thus, Nox4 is an O2 sensor in skeletal muscle, and O2-coupled hydrogen peroxide production by Nox4 governs the redox state of regulatory RyR1 thiols and thereby governs muscle performance. These findings reveal a molecular mechanism for O2-based signaling by an NADPH oxidase and demonstrate a physiological role for oxidative modification of RyR1.

Sun, Qi-An; Hess, Douglas T.; Nogueira, Leonardo; Yong, Sandro; Bowles, Dawn E.; Eu, Jerry; Laurita, Kenneth R.; Meissner, Gerhard; Stamler, Jonathan S.

2011-01-01

322

Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4.  

PubMed

Physiological sensing of O(2) tension (partial O(2) pressure, pO(2)) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO(2) to altered calcium release through the ryanodine receptor-Ca(2+)-release channel (RyR1). Reactive oxygen species are generated in proportion to pO(2) by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca(2+) release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle. Thus, Nox4 is an O(2) sensor in skeletal muscle, and O(2)-coupled hydrogen peroxide production by Nox4 governs the redox state of regulatory RyR1 thiols and thereby governs muscle performance. These findings reveal a molecular mechanism for O(2)-based signaling by an NADPH oxidase and demonstrate a physiological role for oxidative modification of RyR1. PMID:21896730

Sun, Qi-An; Hess, Douglas T; Nogueira, Leonardo; Yong, Sandro; Bowles, Dawn E; Eu, Jerry; Laurita, Kenneth R; Meissner, Gerhard; Stamler, Jonathan S

2011-09-06

323

Endothelin signalling regulates volume-sensitive Cl? current via NADPH oxidase and mitochondrial reactive oxygen species  

PubMed Central

Aims We assessed regulation of volume-sensitive Cl? current (ICl,swell) by endothelin-1 (ET-1) and characterized the signalling pathway responsible for its activation in rabbit atrial and ventricular myocytes. Methods and results ET-1 elicited ICl,swell under isosmotic conditions. Outwardly rectified Cl? current was blocked by the ICl,swell-selective inhibitor DCPIB or osmotic shrinkage and involved ETA but not ETB receptors. ET-1-induced current was abolished by inhibiting epidermal growth factor receptor (EGFR) kinase or phosphoinositide-3-kinase (PI-3K), indicating that these kinases were downstream. Regarding upstream events, activation of ICl,swell by osmotic swelling or angiotensin II (AngII) was suppressed by ETA blockade, whereas AngII AT1 receptor blockade failed to alter ET-1-induced current. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) stimulate ICl,swell. As expected, blockade of NOX suppressed ET-1-induced ICl,swell, but blockade of mitochondrial ROS production with rotenone also suppressed ICl,swell. ICl,swell was activated by augmenting complex III ROS production with antimycin A or diazoxide; in this case, ICl,swell was insensitive to NOX inhibitors, indicating that mitochondria were downstream from NOX. ROS generation in HL-1 cardiomyocytes measured by flow cytometry confirmed the electrophysiological findings. ET-1-induced ROS production was inhibited by blocking either NOX or mitochondrial complex I, whereas complex III-induced ROS production was insensitive to NOX blockade. Conclusion ET-1–ETA signalling activated ICl,swell via EGFR kinase, PI-3K, and NOX ROS production, which triggered mitochondrial ROS production. ETA receptors were downstream effectors when ICl,swell was elicited by osmotic swelling or AngII. These data suggest that ET-1-induced ROS-dependent ICl,swell is likely to participate in multiple physiological and pathophysiological processes.

Deng, Wu; Baki, Lia; Baumgarten, Clive M.

2010-01-01

324

Hypoxia-dependent regulation of nonphagocytic NADPH oxidase subunit NOX4 in the pulmonary vasculature.  

PubMed

Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Chronic hypoxia induces vascular remodeling with medial hypertrophy leading to the development of pulmonary hypertension. We screened lung tissue for the expression of NADPH oxidase subunits. NOX1, NOXA1, NOXO1, p22phox, p47phox, p40phox, p67phox, NOX2, and NOX4 were present in mouse lung tissue. Comparing mice maintained for 21 days under hypoxic (10% O(2)) or normoxic (21% O(2)) conditions, an upregulation exclusively of NOX4 mRNA was observed under hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels. In situ hybridization and immunohistological staining for NOX4 in mouse lungs revealed a localization of NOX4 mRNA and protein predominantly in the media of small pulmonary arteries, with increased labeling intensities after chronic exposure to hypoxia. In isolated pulmonary arterial smooth muscle cells (PASMCs), NOX4 was localized primarily to the perinuclear space and its expression levels were increased after exposure to hypoxia. Treatment of PASMCs with siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation of reactive oxygen species. In lungs from patients with idiopathic pulmonary arterial hypertension (IPAH), expression levels of NOX4, which was localized in the vessel media, were 2.5-fold upregulated. These results support an important role for NOX4 in the vascular remodeling associated with development of pulmonary hypertension. PMID:17585072

Mittal, Manish; Roth, Markus; König, Peter; Hofmann, Simone; Dony, Eva; Goyal, Parag; Selbitz, Anne-Christin; Schermuly, Ralph Theo; Ghofrani, Hossein Ardeschir; Kwapiszewska, Grazyna; Kummer, Wolfgang; Klepetko, Walter; Hoda, Mir Ali Reza; Fink, Ludger; Hänze, Jörg; Seeger, Werner; Grimminger, Friedrich; Schmidt, Harald H H W; Weissmann, Norbert

2007-06-21

325

Lysine triggers apoptosis through a NADPH oxidase-dependent mechanism in human renal tubular cells.  

PubMed

Progressive chronic kidney disease (CKD) is common in lysinuric protein intolerance (LPI), a primary inherited aminoaciduria characterized by massive Lysine excretion in urine. However, by which mechanisms Lysine may cause kidney damage to tubule cells is still not understood. This study determined whether Lysine overloading of human proximal tubular cells (HK-2) in culture enhances apoptotic cell loss and its associated mechanisms. Overloading HK-2 with Lysine levels reproducing those observed in urine of patients affected by LPI (10 mM) increased apoptosis (+30%; p < 0.01 vs.C), as well as Bax and Apaf-1 expressions (+30-50% p < 0.05), while downregulated Bcl-2 (-40% p < 0.05). Apoptosis induced by high Lysine was no longer observed after addition of caspase-9 and caspase-3 inhibitors while caspase-8 inhibitor had no protective effect. High Lysine induced elevations in ROS generation and NADPH oxidase subunits mRNAs (p22 (phox) +106 ± 23%, p67 (phox) +108 ± 22% and gp91 (phox) +75 ± 4% p < 0.05-0.01). In addition, the NADPH oxidase inhibitor DPI prevented both ROS production and apoptosis. Treating HK-2 with antioxidants, such as Cysteine and its analog, N-acetyl-L-cysteine (NAC), rescued the HK-2 from apoptosis induced by Lysine. In summary, our data show that high Lysine in vitro increases the permissiveness of proximal tubule kidney cells to apoptosis by triggering a pathway involving NADPH oxidase signaling. This event may represent a key cellular effect in the increasing the susceptibility of human tubular cells to apoptosis when the tubules cope with a high Lysine load. This effect is instrumental to renal damage and disease progression in patients with LPI. PMID:22403019

Verzola, Daniela; Famŕ, Annamaria; Villaggio, Barbara; Di Rocco, Maia; Simonato, Alchiede; D'Amato, Elena; Gianiorio, Fabio; Garibotto, Giacomo

2012-03-09

326

NADPH oxidase is required for the Sensory Plasticity of the Carotid body by Chronic Intermittent Hypoxia  

PubMed Central

Respiratory moto-neuron response to hypoxia is reflex in nature and carotid body sensory receptor constitutes the afferent limb of this reflex. Recent studies showed that repetitive exposures to hypoxia evokes long term facilitation of sensory nerve discharge (sLTF) of the carotid body in rodents exposed to chronic intermittent hypoxia (CIH). Although studies with anti-oxidants suggested the involvement of reactive oxygen species (ROS)-mediated signaling in eliciting sLTF, the source of and the mechanisms associated with ROS generation have not yet been investigated. We tested the hypothesis that ROS generated by NADPH oxidase (NOX) mediate CIH-evoked sLTF. Experiments were performed on ex vivo carotid bodies from rats and mice exposed either to 10 days of CIH or normoxia. Acute repetitive hypoxia evoked a ~12 fold increase in NOX activity in CIH but not in control carotid bodies, and this effect was associated with up-regulation of NOX2 mRNA and protein, which was primarily localized to glomus cells of the carotid body. sLTF was prevented by NOX inhibitors and was absent in mice deficient in NOX2. NOX activation by CIH required 5-HT release and activation of 5-HT2 receptors coupled to PKC signaling. Studies with ROS scavengers revealed that H2O2 generated from O2·? contributes to sLTF. Priming with H2O2 elicited sLTF of carotid bodies from normoxic control rats and mice, similar to that seen in CIH treated animals. These observations reveal a novel role for NOX-induced ROS signaling in mediating sensory plasticity of the carotid body.

Peng, Y.-J.; Nanduri, J.; Yuan, G.; Wang, N.; Deneris, E.; Pendyala, S.; Natarajan, V.; Kumar, G.K.; Prabhakar, N.R.

2009-01-01

327

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  

PubMed Central

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 gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 ?M) and inhibited by diphenyliodonium (5 ?M). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or orresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.

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

1996-01-01

328

Polychlorinated Biphenyls Disrupt Intestinal Integrity via NADPH Oxidase-Induced Alterations of Tight Junction Protein Expression  

PubMed Central

Background Polychlorinated biphenyls (PCBs) are widely distributed environmental toxicants that contribute to numerous disease states. The main route of exposure to PCBs is through the gastrointestinal tract; however, little is known about the effects of PCBs on intestinal epithelial barrier functions. Objective The aim of the present study was to address the hypothesis that highly chlorinated PCBs can disrupt gut integrity at the level of tight junction (TJ) proteins. Methods Caco-2 human colon adenocarcinoma cells were exposed to one of the following PCB congeners: PCB153, PCB118, PCB104, and PCB126. We then assessed NAD(P)H oxidase (NOX) activity and expression and the barrier function of Caco-2 cells. In addition, the integrity of intestinal barrier function and expression of TJ proteins were evaluated in C57BL/6 mice exposed to individual PCBs by oral gavage. Results Exposure of Caco-2 cells to individual PCB congeners resulted in activation of NOX and increased permeability of fluorescein isothiocyanate (FITC)-labeled dextran (4 kDa). Treatment with PCB congeners also disrupted expression of TJ proteins zonula occludens-1 (ZO-1) and occludin in Caco-2 cells. Importantly, inhibition of NOX by apocynin significantly protected against PCB-mediated increase in epithelial permeability and alterations of ZO-1 protein expression. Exposure to PCBs also resulted in alterations of gut permeability via decreased expression of TJ proteins in an intact physiological animal model. Conclusions These results suggest that oral exposure to highly chlorinated PCBs disrupts intestinal epithelial integrity and may directly contribute to the systemic effects of these toxicants.

Choi, Yean Jung; Seelbach, Melissa J.; Pu, Hong; Eum, Sung Yong; Chen, Lei; Zhang, Bei; Hennig, Bernhard; Toborek, Michal

2010-01-01

329

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.

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

2012-01-01

330

Antimutagenic activity of oxidase enzymes  

SciTech Connect

By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity.

Agabeili, R.A.

1986-11-01

331

Effects of NADPH oxidase inhibitor apocynin on real-time blood hydrogen peroxide release in femoral artery\\/vein ischemia and reperfusion  

Microsoft Academic Search

Background: Vascular endothelial dysfunction can initiate oxidative stress during ischemiaJreperfusion (IIR). Endothelial dysfunction is characterized by an increase in blood hydrogen peroxide (H20 ]) and a decrease in endothelial-derived nitric oxide (NO) bioavailability. Previous studies using Go 6983, a broad-spectrum protein kinase C inhibitor that can inhibit NADPH oxidase activity, has attenuated blood H20 ] levels during femoralliR in vivo.

C Woodsworth Parker; Kyle D. Bartol; Kerry-Anne Perkins; ChenQian; YoungLindonH

2012-01-01

332

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

Microsoft Academic Search

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-?B (NF-?B) controls the expression of many genes associated to inflammation-related diseases, in this study we have investigated the role of NF-?B signaling in the regulation of Nox1 and Nox4 transcription in

Adrian Manea; Laurentia I. Tanase; Monica Raicu; Maya Simionescu

2010-01-01

333

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

Microsoft Academic Search

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

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

1997-01-01

334

Modulation of chronic hypoxia-induced chemoreceptor hypersensitivity by NADPH oxidase subunits in rat carotid body  

PubMed Central

Previous studies in our laboratory established that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) facilitate the open state of a subset of K+ channels in oxygen-sensitive type I cells of the carotid body. Thus pharmacological inhibition of NOX or deletion of a NOX gene resulted in enhanced chemoreceptor sensitivity to hypoxia. The present study tests the hypothesis that chronic hypoxia (CH)-induced hypersensitivity of chemoreceptors is modulated by increased NOX activity and elevated levels of ROS. Measurements of dihydroethidium fluorescence in carotid body tissue slices showed that increased ROS production following CH (14 days, 380 Torr) was blocked by the specific NOX inhibitor 4-(2-amino-ethyl)benzenesulfonyl fluoride (AEBSF, 3 ?M). Consistent with these findings, in normal carotid body AEBSF elicited a small increase in the chemoreceptor nerve discharge evoked by an acute hypoxic challenge, whereas after 9 days of CH the effect of the NOX inhibitor was some threefold larger (P < 0.001). Evaluation of gene expression after 7 days of CH showed increases in the isoforms NOX2 (?1.5-fold) and NOX4 (?3.8-fold) and also increased presence of the regulatory subunit p47phox (?4.2-fold). Involvement of p47phox was further implicated in studies of isolated type I cells that demonstrated an ?8-fold and an ?11-fold increase in mRNA after 1 and 3 days, respectively, of hypoxia in vivo. These findings were confirmed in immunocytochemical studies of carotid body tissue that showed a robust increase of p47phox in type I cells after 14 days of CH. Our findings suggest that increased ROS production by NOX enzymes in type I cells dampens CH-induced hypersensitivity in carotid body chemoreceptors.

He, L.; Liu, X.; Chen, J.; Stensaas, L.; Fidone, S.

2010-01-01

335

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.

Quinn, Mark T.; Schepetkin, Igor A.

2009-01-01

336

NADPH oxidase-mediated upregulation of connexin43 contributes to podocyte injury.  

PubMed

The gap junction protein connexin43 (Cx43) was markedly increased in podocytes in a rat model of nephrosis induced by puromycin. However, the mechanisms and roles of the altered Cx43 in podocytes are still unclear. Given that oxidative stress mediates podocyte injury under a variety of pathological situations, we examined the possible involvement of an oxidative stress-related mechanism in the regulation of Cx43. Incubation of podocytes with puromycin led to a time- and concentration-dependent loss of cell viability, which was preceded by an elevation in Cx43 levels. Concomitantly, puromycin also induced NOX4 expression and promoted superoxide (O(2)(·-)) generation. Inhibition of NADPH oxidase with apocynin and diphenyleneiodonium chloride or addition of the superoxide dismutase mimetic tempol completely abrogated, whereas the O(2)(·-) donors menadione and 2,3-dimethoxy-1,4-naphthoquinone reproduced, the effects of puromycin on Cx43 expression and cell injury. Further analysis demonstrated that treatment of podocytes with several structurally different gap-junction inhibitors significantly attenuated the cytotoxicity of puromycin. Our results thus indicate that NADPH oxidase-mediated upregulation of Cx43 contributes to podocyte injury. PMID:22824863

Yan, Qiaojing; Gao, Kun; Chi, Yuan; Li, Kai; Zhu, Ying; Wan, Yigang; Sun, Wei; Matsue, Hiroyuki; Kitamura, Masanori; Yao, Jian

2012-07-21

337

Apocynin, an NADPH oxidase inhibitor, suppresses progression of prostate cancer via Rac1 dephosphorylation.  

PubMed

Recently, considerable evidence has been generated that oxidative stress contributes to the etiology and pathogenesis of prostate cancer. The present study focused on the effects of apocynin, an inhibitor of the NADPH oxidase which generates intracellular superoxide, on a rat androgen-independent prostate cancer cell line (PLS10) in vitro and in vivo. Apocynin significantly inhibited cell proliferation of PLS10 cells via G1 arrest of the cell cycle in vitro. Surprisingly, it did not affect reactive oxygen species (ROS) but inhibited phosphorylation of Rac1, one component of the NADPH oxidase complex. A Rac1 inhibitor, NSC23766, also inhibited cell proliferation, and both apocynin and NSC23766 reduced phosphorylation of Rac1 and NF-?B, as well as cyclin D1. Furthermore, in a xenograft model of prostate cancer with PLS10, apocynin suppressed tumor growth and metastasis in a dose dependent manner in vivo, with reduction of cell proliferation and vessel number in the tumors. Expression and secretion of vascular endothelial growth factor (VEGF) were reduced by apocynin treatment in vivo and in vitro, respectively. In conclusion, despite no apparent direct relationship with oxidative stress, apocynin inhibited growth of androgen-independent prostate cancer in vitro and in vivo. Apocynin thus warrants further attention as a potential anti-tumor drug. PMID:23664830

Suzuki, Shugo; Pitchakarn, Pornsiri; Sato, Shinya; Shirai, Tomoyuki; Takahashi, Satoru

2013-05-09

338

Purification of human neutrophil NADPH oxidase cytochrome b-558 and association with Rap 1A.  

PubMed

The availability of sufficient quantities of highly purified phagocyte cytochrome b-558 has been necessary for many of the biochemical and immunological analyses of this important NADPH oxidase component, and it was only through the analysis of highly purified cytochrome b that the subunit composition was elucidated and the small subunit (p22-phox) was cloned and sequenced. In addition, the association of the small GTP-binding protein Rap1A with cytochrome b-558 was discovered through the analysis of purified cytochrome b. The procedures described here provide an easy, efficient, and highly reproducible method for the purification of cytochrome b as well as cytochrome b-Rap1A complexes. The ability to purify cytochrome b and cytochrome b-Rap1A complexes will also allow further analysis of the structure of this novel plasma membrane redox protein and the role of its association with low molecular weight GTP-binding proteins in the structure and regulation of the phagocyte NADPH oxidase. PMID:8524135

Quinn, M T; Parkos, C A; Jesaitis, A J

1995-01-01

339

Cigarette smoke extract upregulates heme oxygenase-1 via PKC/NADPH oxidase/ROS/PDGFR/PI3K/Akt pathway in mouse brain endothelial cells  

PubMed Central

Background In the brain, the inducible form of heme oxygenase (HO-1) has been recently demonstrated to exacerbate early brain injury produced by intracerebral hemorrhagic stroke which incident rate has been correlated with cigarette smoking previously. Interestingly, cigarette smoke (CS) or chemicals present in CS have been shown to induce HO-1 expression in various cell types, including cerebral endothelial cells. However, the mechanisms underlying CS modulating HO-1 protein expression are not completely understood in the brain vessels. Objective The aim of the present study was to investigate the mechanisms underlying CS modulating HO-1 protein expression in cerebral endothelial cells. Methods Cultured cerebral endothelial cells (bEnd.3) were used to investigate whether a particulate phase of cigarette smoke extract (PPCSE) regulates HO-1 expression and to investigate the molecular mechanisms involved in HO-1 expression in bEnd.3 cells. Results We demonstrated that PPCSE (30 ?g/ml) significantly induced HO-1 protein expression and its enzymatic activity in bEnd.3 cells determined by western blotting and bilirubin formation, respectively. PPCSE-induced HO-1 expression was mediated through phosphatidylcholine phospholipase C (PC-PLC), PKC?, and PI3K/Akt which were observed by pretreatment with their respective pharmacological inhibitors or transfection with dominant negative mutants of PKC? and Akt. ROS scavenger (N-acetyl-L-cysteine, NAC) blocked the PPCSE-induced ROS generation and HO-1 expression. Pretreatment with selective inhibitors of PKC? (rottlerin) and NADPH oxidase [diphenyleneiodonium chloride (DPI) and apocynin (APO)] attenuated the PPCSE-induced NADPH oxidase activity, ROS generation, and HO-1 expression. In addition, we found that PPCSE induced PI3K/Akt activation via NADPH oxidase/ROS-dependent PDGFR phosphorylation. Conclusions Taken together, these results suggested that PPCSE-induced HO-1 expression is mediated by a PC-PLC/PKC?/NADPH oxidase-dependent PDGFR/PI3K/Akt pathway in bEnd.3 cells.

2011-01-01

340

AAV-Based RNAi Silencing of NADPH Oxidase gp91phox Attenuates Cold-Induced Cardiovascular Dysfunction  

PubMed Central

Abstract Clinical observations and epidemiological surveys indicated that the prevalence of hypertension and heart diseases is increased in cold regions or during winter. Cold exposure increased NADPH oxidase gp91phox protein expression in heart, kidneys, and aorta in rats. The aim of this study was to investigate if RNA interference (RNAi) silencing of gp91phox would attenuate cold-induced hypertension and cardiovascular and renal damage. The recombinant adeno-associated virus serotype 2 (AAV-2) vector carrying gp91phox-shRNA (gp91-shRNA) was constructed for inhibiting gp91phox protein expression in cold-exposed rats. Blood pressure (BP) was monitored using a telemetry system. BP was increased in the Control-shRNA and PBS groups within 1 week of exposure to moderate cold (5°C) and reached a plateau after 7 weeks. The cold-induced increase in BP was attenuated significantly by intravenous delivery of gp91-shRNA (1.25×1010 particles/rat, 0.5?mL). One single dose of gp91-shRNA controlled hypertension for up to 10 weeks. In addition, gp91-shRNA reversed cold-induced vascular dysfunction. gp91-shRNA abolished the cold-induced up-regulation of gp91phox protein expression in heart, kidneys, and aorta, confirming effective silencing of gp91phox. The cold-induced increases in NADPH oxidase activity and superoxide production were eliminated by silencing of gp91phox, suggesting that the cold-induced up-regulation of NADPH oxidase activity may be attributed to the increased gp91phox protein expression. RNAi silencing of gp91phox abolished cold-induced cardiac and renal hypertrophy and attenuated aortic, coronary, and renal remodeling. The up-regulation of gp91phox may play a critical role in cold-induced cardiovascular dysfunction and organ damage. AAV delivery of gp91-shRNA may be a new and effective therapeutic approach for cold-related cardiovascular disorders.

Wang, Xiuqing; Skelley, Lucille; Wang, Bo; Mejia, Ayesha; Sapozhnikov, Val

2012-01-01

341

The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells  

Microsoft Academic Search

The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells

Junya Kuroda; Kazunori Nakagawa; Tomoko Yamasaki; Kei-ichiro Nakamura; Ryu Takeya; Futoshi Kuribayashi; Shinobu Imajoh-Ohmi; Kazuhiko Igarashi; Yosaburo Shibata; Katsuo Sueishi; Hideki Sumimoto

2005-01-01

342

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.

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

343

Increased expression of NAD(P)H oxidase subunit p67phox in the renal medulla contributes to excess oxidative stress and salt-sensitive hypertension  

PubMed Central

SUMMARY NAD(P)H oxidase has been shown to be important in the development of salt-sensitive hypertension. Here we show that the expression of a subunit of NAD(P)H oxidase, p67phox, was increased in response to a high salt diet in the outer renal medulla of the Dahl salt-sensitive (SS) rat, an animal model for human salt-sensitive hypertension. The higher expression of p67phox, not the other subunits observed, was associated with higher NAD(P)H oxidase activity and salt-sensitivity in SS rats compared with a salt-resistant strain. Genetic mutations of the SS allele of p67phox were found in the promoter region and contributed to higher promoter activity than that of the salt-resistant strain. To verify the importance of p67phox, we disrupted p67phox in SS rats using zinc finger nucleases technology. These rats exhibited a significant reduction of salt-sensitive hypertension and renal medullary oxidative stress and injury. p67phox could represent a target for salt-sensitive hypertension therapy.

Feng, Di; Yang, Chun; Geurts, Aron; Kurth, Terry; Liang, Mingyu; Lazar, Jozef; Mattson, Dave; O'Connor, Paul; Cowley, Allen W.

2012-01-01

344

Platelet particle formation by anti GPIIIa49-66 Ab, Ca2+ ionophore A23187, and phorbol myristate acetate is induced by reactive oxygen species and inhibited by dexamethasone blockade of platelet phospholipase A2, 12-lipoxygenase, and NADPH oxidase.  

PubMed

An HIV antibody (Ab) against platelet integrin GPIIIa49-66 induces complement-independent platelet particle formation by the elaboration of reactive oxygen species (ROS) downstream of the activation of the platelet NADPH oxidase by the 12-lipoxygenase (12-LO) product 12(S)-HETE. To determine whether other inducers of platelet particle formation also function via the induction of ROS, we examined the effects of the Ca(2+) ionophore A23187 and phorbol myristate acetate (PMA). Both agents induced oxidative platelet particle formation in an identical fashion as Ab, requiring Ca(2+) flux and 12(S)-HETE production as well as intact NADPH oxidase and 12-LO pathways. Since HIV-ITP patients with this Ab correct their platelet counts with dexamethasone (Dex), we examined the role of this steroid in this unique autoimmune disorder. Dex at therapeutic concentrations inhibited Ab-, A23187-, or PMA-induced platelet particle formation by inhibiting platelet PLA(2), 12-LO, and NADPH oxidase. The operational requirement of translocation of PLA(2), 12-LO, and NADPH oxidase components (p67 phox) from cytosol to membrane for induction of ROS was both inhibited and partially reversed by Dex in platelets. We conclude that (1) platelet particle formation can be induced by the generation of ROS; and (2) platelet PLA(2), 12-LO, NADPH oxidase, and cytosol membrane translocation, requirements for ROS production, are inhibited by Dex. PMID:17545506

Nardi, Michael A; Gor, Yelena; Feinmark, Steven J; Xu, Fang; Karpatkin, Simon

2007-06-01

345

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,