scholarly journals Rac Activation Induces NADPH Oxidase Activity in Transgenic COSphoxCells, and the Level of Superoxide Production Is Exchange Factor-dependent

2002 ◽  
Vol 277 (21) ◽  
pp. 19220-19228 ◽  
Author(s):  
Marianne O. Price ◽  
Simon J. Atkinson ◽  
Ulla G. Knaus ◽  
Mary C. Dinauer
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Superoxide Production ◽  
Nadph Oxidase Activity ◽  
Exchange Factor

Palmitate increases superoxide production through mitochondrial electron transport chain and NADPH oxidase activity in skeletal muscle cells

2008 ◽  
Vol 216 (3) ◽  
pp. 796-804 ◽  
Author(s):  
Rafael Herling Lambertucci ◽  
Sandro Massao Hirabara ◽  
Leonardo dos Reis Silveira ◽  
Adriana Cristina Levada‐Pires ◽  
Rui Curi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Electron Transport ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Electron Transport Chain ◽  
Muscle Cells ◽  
Superoxide Production ◽  
Mitochondrial Electron Transport Chain ◽  
Nadph Oxidase Activity ◽  
Transport Chain

scholarly journals FcγR-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

Blood ◽  
2008 ◽  
Vol 112 (9) ◽  
pp. 3867-3877 ◽  
Author(s):  
Wei Tian ◽  
Xing Jun Li ◽  
Natalie D. Stull ◽  
Wenyu Ming ◽  
Chang-Il Suh ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Superoxide Production ◽  
Resting Cells ◽  
Cellular Activation ◽  
Nadph Oxidase Activity ◽  
Phagocyte Nadph Oxidase ◽  
Before And After ◽  
Membrane Bound

AbstractThe phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after phagosome internalization. We conclude that p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.

Arachidonic Acid Rescues Defects on PMA-Elicited NADPH Oxidase Activity in Rac2-Deficient Neutrophils.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2386-2386
Author(s):  
Chaekyun Kim ◽  
Mary C. Dinauer
Keyword(s):  
Arachidonic Acid ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Actin Polymerization ◽  
Rho Gtpase ◽  
Superoxide Production ◽  
Wild Type ◽  
Functional Responses ◽  
Nadph Oxidase Activity ◽  
Nadph Oxidase Activation

Abstract Rac2 is a hematopoietic-specific Rho-GTPase implicated as an important constituent of the NADPH oxidase complex. We previously showed that Rac2 plays a stimulus-specific role in regulating NADPH oxidase activation and other functional responses in neutrophils [Kim and Dinauer, JI 166, 2001]. Here we investigate the effect of arachidonic acid (AA) on Rac2-regulated NADPH oxidase activity. Superoxide production in rac2-/- neutrophils was significantly lower (~4-fold) than that of wild-type when stimulated with PMA or AA alone. However, exogenously added AA (10 μM) fully restored the defect in PMA-elicited NADPH oxidase activity in rac2−/ − neutrophils, while having no effect on FMLP-elicited superoxide production. Impaired PMA- or AA-induced F-actin polymerization in rac2−/ − neutrophils was also not restored by co-stimulation with PMA and AA. Taken together, these observations suggest that there are agonist- and pathway-specific differences in the underlying basis of functional defects in rac2−/ − neutrophils. To further investigate possible mechanisms of AA-mediated rescue of PMA-stimulated NADPH oxidase activation in rac2−/ − neutrophils, we measured protein expression and activity of cytosolic phospholipase A2 (cPLA2) and protein kinase C (PKC). The expression of cPLA2 and PMA-stimulated release of AA was similar between wild-type and rac2−/ − neutrophils, suggesting that defects in AA production by PMA-stimulated rac2−/ − neutrophils do not account for the effect of exogenous AA on oxidase activity. The neutrophil expression of PKC isoforms (α, β, δ, ζ) was also similar between genotypes. The cytosolic p47phox and p67phox components of NADPH oxidase were translocated to the plasma membrane upon stimulation with PMA in both genotypes, and no additional translocation in either wild-type or rac2−/ − neutrophils was detected upon co-stimulation with AA. The level of activated Rac1-GTP was similar between genotypes following stimulation, and was not increased by co-stimulation with PMA and AA. These studies indicate that the addition of exogenous AA reconstitutes PMA-elicited superoxide production in rac2−/ − neutrophils independent of the effects on translocation of p47phox and p67phox and activation of Rac1 GTPase. We hypothesize that the effect of AA is exerted through conformational changes of the assembled NADPH oxidase.

The Phosphoinositide-Binding Protein p40phox Regulates NADPH Oxidase Activation Rather Than Assembly during FcγIIA Receptor-Induced Phagocytosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 678-678 ◽  
Author(s):  
Wei Tian ◽  
Xing Jun Li ◽  
Natalie D. Stull ◽  
Chang-Il Suh ◽  
Sergio Grinstein ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Superoxide Production ◽  
Null Mouse ◽  
Target Sequence ◽  
Resting Cells ◽  
Nadph Oxidase Activity ◽  
Px Domain ◽  
Significant Difference ◽  
Nadph Oxidase Activation

Abstract Many critical features of the organization and regulation of the phagocyte NADPH oxidase, a complex multi-subunit enzyme that generates superoxide for microbial killing, remain poorly defined. The active enzyme includes a membrane-bound flavocytochrome b along with p47phox, p67phox, p40phox, and Rac-GTP that are present in the cytosol of resting cells. p67phox is linked by high affinity interactions with both p47phox and p40phox, which appear to translocate as a trimeric complex upon cellular activation. The p47phox subunit acts as an adaptor to promote translocation by docking at a proline-rich target sequence on the flavocytochrome, and p67phox is a Rac-GTP effector containing a domain that activates electron transport. In contrast, the function of p40phox, which is not required for high level oxidase activity in cell free systems, is poorly understood. Recently, our group showed that p40phox plays key role in the activation of superoxide production during phagocytosis of IgG-opsonized targets in COSphoxFcγR cells. This model cell line contains stable transgenes for the flavocytochrome, p47phox, p67phox, and the FcγIIA receptor, without or with an additional transgene for p40phox. p40phox-dependent coupling of FcγR-mediated phagocytosis to superoxide production required an intact p40phox PX domain, which binds to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide generated by class III PI3 kinases in phagosome membranes (Suh et al J Exp Med 203, 1915Suh et al J Exp Med 203, 2006). Furthermore, a newly developed p40phox-null mouse exhibits reduced neutrophil NADPH oxidase activity in response to selected agonists, including IgG-opsonized targets (Ellson et al J Exp Med 203, 1927Ellson et al J Exp Med 203, 2006). In the current study, we investigated whether p40phox is required for translocation of p67phox during phagocytosis. We generated COSphoxFcγR cells expressing YFP-tagged p67phox from a stable transgene instead of untagged p67phox. Following incubation with IgG-opsonized sheep red blood cells (IgG-RBC), p67phox was detected on phagosome membranes at both early stages of phagosome cup formation and after closure, independent of whether or not p40phox was also co-expressed. However, NADPH oxidase activity was not detected in IgG-RBC phagosomes in COSphoxFcγR-p67phox-YFP cells unless p40phox was present. PMA-activated superoxide production was independent of p40phox, and Western blotting indicated there was no significant difference in expression of the other oxidase subunits in COSphoxFcγR-p67phox-YFP cells without or with the p40phox transgene. Further studies in PLB-985 granulocytes expressing stable transgenes for either YFP-tagged p67phox or p40phox showed that the PI3K inhibitor wortmannin inhibited phagosome NADPH oxidase activity and translocation of p40phox, but localization of p67phox to phagosomes was unaffected. These results indicate that although p40phox positively regulates NADPH oxidase activation during phagocytosis, recruitment of p67phox to the phagosome is independent of p40phox. Taken together, these data suggest that the PX domain of p40phox acts as a PI3P-dependent switch to activate the membrane-assembled NADPH oxidase complex.

scholarly journals 20-HETE increases NADPH oxidase-derived ROS production and stimulates the L-type Ca2+ channel via a PKC-dependent mechanism in cardiomyocytes

2010 ◽  
Vol 299 (4) ◽  
pp. H1109-H1117 ◽  
Author(s):  
Qinghua Zeng ◽  
Yong Han ◽  
Yuyan Bao ◽  
Wei Li ◽  
Xingting Li ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Molecular Mechanism ◽  
Oxidase Activity ◽  
Ischemia Reperfusion ◽  
Oxidase Inhibitor ◽  
Superoxide Production ◽  
Patch Clamp Recording ◽  
Stimulatory Action ◽  
Nadph Oxidase Activity ◽  
Dependent Mechanism

The production of 20-hydroxyeicosatetraenoic acid (20-HETE) is increased during ischemia-reperfusion, and inhibition of 20-HETE production has been shown to reduce infarct size caused by ischemia. This study was aimed to discover the molecular mechanism underlying the action of 20-HETE in cardiac myocytes. The effect of 20-HETE on L-type Ca2+ currents ( ICa,L) was examined in rat isolated cardiomyocytes by patch-clamp recording in the whole cell mode. Superfusion of cardiomyocytes with 20-HETE (10–100 nM) resulted in a concentration-dependent increase in ICa,L, and this action of 20-HETE was attenuated by a specific NADPH oxidase inhibitor, gp91ds-tat (5 μM), or a superoxide scavenger, polyethylene glycol-superoxide dismutase (25 U/ml), suggesting that NADPH-oxidase-derived superoxide is involved in the stimulatory action of 20-HETE on ICa,L. Treatment of cardiomyocytes with 20-HETE (100 nM) increased both NADPH oxidase activity and superoxide production by approximately twofold. To study the molecular mechanism mediating the 20-HETE-induced increase in NADPH oxidase activity, PKC activity was measured in cardiomyocytes. Incubation of the cells with 20-HETE (100 nM) significantly increased PKC activity, and pretreatment of cardiomyocytes with a selective PKC inhibitor, GF-109203 (1 μM), attenuated the 20-HETE-induced increases in ICa,L and in NADPH oxidase activity. In summary, 20-HETE stimulates NADPH oxidase-derived superoxide production, which activates L-type Ca2+ channels via a PKC-dependent mechanism in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of cardiac ischemic diseases.

scholarly journals Regulation of NADPH Oxidase-Mediated Superoxide Production by Acetylation and Deacetylation

2021 ◽  
Vol 12 ◽  
Author(s):  
Ning Xia ◽  
Stefan Tenzer ◽  
Oleg Lunov ◽  
Martin Karl ◽  
Thomas Simmet ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Trichostatin A ◽  
Oral Treatment ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Superoxide Production ◽  
Sirtuin 1 ◽  
Binding Potential ◽  
Free System ◽  
Nadph Oxidase Activity

Oral treatment of apolipoprotein E-knockout (ApoE-KO) mice with the putative sirtuin 1 (SIRT1) activator resveratrol led to a reduction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the heart. In contrast, the SIRT1 inhibitor EX527 enhanced the superoxide production in isolated human polymorphonuclear granulocytes. In human monocytic THP-1 cells, phorbol ester-stimulated superoxide production was enhanced by inhibitors of histone deacetylases (HDACs; including quisinostat, trichostatin A (TSA), PCI34051, and tubastatin A) and decreased by inhibitors of histone acetyltransferases [such as garcinol, curcumin, and histone acetyltransferase (HAT) Inhibitor II]. These results indicate that protein acetylation and deacetylation may represent crucial mechanisms regulating NADPH oxidase-mediated superoxide production. In cell-free systems, incubation of recombinant Rac1 with SIRT1 resulted in decreased Rac1 acetylation. Mass spectrometry analyses identified lysine 166 (K166) in Rac1 as a residue targeted by SIRT1. Deacetylation of Rac1 by SIRT1 markedly reduced the interaction of Rac1 with p67phox in in vitro assays. Computational modeling analyses revealed that K166 deacetylation of Rac1 led to a 5-fold reduction in its binding affinity to guanosine-5'-triphosphate, and a 21-fold decrease in its binding potential to p67phox. The latter is crucial for Rac1-mediated recruitment of p67phox to the membrane and for p67phox activation. In conclusion, both SIRT1 and non-sirtuin deacetylases play a role in regulating NADPH oxidase activity. Rac1 can be directly deacetylated by SIRT1 in a cell-free system, leading to an inhibition of Rac1-p67phox interaction. The downstream targets of non-sirtuin deacetylases are still unknown. The in vivo significance of these findings needs to be investigated in future studies.

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