Validation of Lucigenin (Bis-N-methylacridinium) as a Chemilumigenic Probe for Detecting Superoxide Anion Radical Production by Enzymatic and Cellular Systems

Li, Yunbo; Zhu, Hong; Kuppusamy, Periannan; Roubaud, Valérie; Zweíer, Jay L.; Trush, Michael A.

doi:10.1074/jbc.273.4.2015

Cited by 501 publications

(291 citation statements)

References 27 publications

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“…Different times of mitochondrial incubation (from 5 to 20 min) were tested. Because enzymatic systems that produce superoxide can lead to oxygen consumption (Li et al 1998), the incubation of the amoeba mitochondria with xanthine/xanthine oxidase was performed in an open chamber in which the medium was constantly stirred to balance the air saturation. For our experimental conditions, we confirmed the xanthine concentration-dependent exogenous superoxide generation using the nitroblue tetrazolium (NBT) reduction test (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Hydroxynonenal, a lipid peroxidation end product, stimulates uncoupling protein activity in Acanthamoeba castellanii mitochondria; the sensitivity of the inducible activity to purine nucleotides depends on the membranous ubiquinone redox state

Woyda-Płoszczyca

Jarmuszkiewicz

2012

J Bioenerg Biomembr

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We studied the influence of exogenously generated superoxide and exogenous 4-hydroxy-2-nonenal (HNE), a lipid peroxidation end product, on the activity of the Acanthamoeba castellanii uncoupling protein (AcUCP). The superoxide-generating xanthine/xanthine oxidase system was insufficient to induce mitochondrial uncoupling. In contrast, exogenously added HNE induced GTP-sensitive AcUCP-mediated mitochondrial uncoupling. In nonphosphorylating mitochondria, AcUCP activation by HNE was demonstrated by increased oxygen consumption accompanied by a decreased membrane potential and ubiquinone (Q) reduction level. The HNE-induced GTP-sensitive proton conductance was similar to that observed with linoleic acid. In phosphorylating mitochondria, the HNEinduced AcUCP-mediated uncoupling decreased the yield of oxidative phosphorylation. We demonstrated that the efficiency of GTP to inhibit HNE-induced AcUCPmediated uncoupling was regulated by the endogenous Q redox state. A high Q reduction level activated AcUCP by relieving the inhibition caused by GTP while a low Q reduction level favoured the inhibition. We propose that the regulation of UCP activity involves a rapid response through the endogenous Q redox state that modulates the inhibition of UCP by purine nucleotides, followed by a late response through lipid peroxidation products resulting from an increase in the formation of reactive oxygen species that modulate the UCP activation.

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Section: Resultsmentioning

confidence: 99%

Hydroxynonenal, a lipid peroxidation end product, stimulates uncoupling protein activity in Acanthamoeba castellanii mitochondria; the sensitivity of the inducible activity to purine nucleotides depends on the membranous ubiquinone redox state

Woyda-Płoszczyca

Jarmuszkiewicz

2012

J Bioenerg Biomembr

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“…Measurements on intact arteries with lucigenin at 5 mol/L have been corroborated with electron spin resonance and were not complicated by its redox cycling. 29 As shown in Figure 5A, O 2 ⅐ Ϫ was significantly elevated in aortas isolated from a CD40L-exposed mouse compared with aortas in sham-treated mice (nϭ9; PϽ0.05, sham versus CD40L-treated mice). In hSOD-TG mice, basal aortic O 2 ⅐ Ϫ release was significantly lower than in C57BL6 mice (Figure 5A; nϭ7; PϽ0.05, C57BL6 versus hSOD-TG).…”

Section: Onoo ؊ -Dependent Tyrosine Nitration Of Pgis Is Operated In mentioning

confidence: 94%

CD40 Ligand–Dependent Tyrosine Nitration of Prostacyclin Synthase In Vivo

Davis

Zou

2005

Circulation

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Background-Cells in human atherosclerotic lesions express the immune mediator CD40 and its ligand, CD40L, but the mechanisms and the mediators by which CD40L contributes to atherosclerosis are poorly defined. Here, we show how CD40L increases vascular inflammation and thrombosis via tyrosine nitration and inhibition of prostacyclin synthase (PGIS), an enzyme with antithrombotic, antiproliferative, and dilatory functions in the normal vasculature. Methods and Results-Exposure of cultured human aortic endothelial cells to clinically relevant concentrations of CD40L (20 to 80 ng/mL) dose-dependently increased the production of superoxide (O 2 ⅐ Ϫ ), decreased nitric oxide (NO) bioactivity, and increased PGIS nitration. Furthermore, inhibition of CD40 expression by small interfering RNA blocked the effects of CD40L on O 2 ⅐ Ϫ , NO bioactivity, and PGIS nitration, which indicates a specific effect of CD40L. In addition, either depletion of mitochondria ( 0 cells, ie, mitochondria-depleted cells, to prevent mitochondrial O 2 ⅐ Ϫ ) or adenoviral overexpression of superoxide dismutase, as well as inhibition of NO synthase, abolished the CD40L-enhanced PGIS nitration, which implies that the mitochondria might be the source of O 2 ⅐ Ϫ and thus peroxynitrite (ONOO Ϫ ). Furthermore, SQ29548, a thromboxane A 2 /prostaglandin H 2 receptor antagonist, significantly reduced CD40L-enhanced expression of intercellular adhesion molecule-1. Finally, administration of CD40L resulted in PGIS inhibition and nitration in the aortas of C57BL6 mice but less in mice overexpressing human superoxide dismutase, which suggests that ONOO Ϫ might be required for CD40L-enhanced PGIS nitration in vivo. Conclusions-We

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“…Luciferase activity was assessed at 6 h after cytokine treatment (unless otherwise specified) by using 5 mg of cell lysate. NADPH oxidase activities were analyzed by measuring the rate of O 2 -generation with a chemiluminescent, lucigenin-based assay (35), as previously applied to vesicles (32). In brief, 5 mM lucigenin (Sigma-Aldrich) in PBS was incubated with vesicular fractions for 10 min in the dark, and reactions were initiated by addition of 100 mM NADPH (Sigma-Aldrich).…”

Section: Cytokine Treatments and Vesicular Isolationmentioning

confidence: 99%

Endosomal Nox2 Facilitates Redox-Dependent Induction of NF-κB by TNF-α

Spencer

Oakley

et al. 2009

Antioxidants & Redox Signaling

110

133

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Growing evidence suggests that NADPH oxidase (Nox)-derived reactive oxygen species (ROS) play important roles in regulating cytokine signaling. We have explored how TNF-a induction of Nox-dependent ROS influences NF-kB activation. Cellular stimulation by TNF-a induced NADPH-dependent superoxide production in the endosomal compartment, and this ROS was required for IKK-mediated activation of NF-kB. Inhibiting endocytosis reduced the ability of TNF-a to induce both NADPH-dependent endosomal superoxide and NF-kB, supporting the notion that redox-dependent signaling of the receptor occurs in the endosome. Molecular analyses demonstrated that endosomal H 2 O 2 was critical for the recruitment of TRAF2 to the TNFR1=TRADD complex after endocytosis. Studies using both Nox2 siRNA and Nox2-knockout primary fibroblasts indicated that Nox2 was critical for TNF-a-mediated induction of endosomal superoxide. Redox-active endosomes that form after TNF-a or IL-1b induction recruit several common proteins (Rac1, Nox2, p67 phox , SOD1), while also retaining specificity for ligand-activated receptor effectors. Our studies suggest that TNF-a and IL-1b signaling pathways both can use Nox2 to facilitate redox activation of their respective receptors at the endosomal level by promoting the redox-dependent recruitment of TRAFs. These studies help to explain how cellular compartmentalization of redox signals can be used to direct receptor activation from the plasma membrane.

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Validation of Lucigenin (Bis-N-methylacridinium) as a Chemilumigenic Probe for Detecting Superoxide Anion Radical Production by Enzymatic and Cellular Systems

Abstract: Lucigenin is most noted for its wide use as a chemilu

Cited by 501 publications

References 27 publications

Hydroxynonenal, a lipid peroxidation end product, stimulates uncoupling protein activity in Acanthamoeba castellanii mitochondria; the sensitivity of the inducible activity to purine nucleotides depends on the membranous ubiquinone redox state

Hydroxynonenal, a lipid peroxidation end product, stimulates uncoupling protein activity in Acanthamoeba castellanii mitochondria; the sensitivity of the inducible activity to purine nucleotides depends on the membranous ubiquinone redox state

CD40 Ligand–Dependent Tyrosine Nitration of Prostacyclin Synthase In Vivo

Endosomal Nox2 Facilitates Redox-Dependent Induction of NF-κB by TNF-α

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