ERO1α primes the ryanodine receptor to respond to arsenite with concentration dependent Ca2+ release sequentially triggering two different mechanisms of ROS formation
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A 6 h exposure of U937 cells to 2.5 μM arsenite stimulates low Ca2+ release from the inositol 1, 4, 5-triphosphate receptor (IP3R), causing a cascade of causally connected events, i.e., endoplasmic reticulum oxidoreductin-1α (ERO1α) expression, activation of the ryanodine receptor (RyR), mitochondrial Ca2+ accumulation, mitochondrial superoxide formation and further ERO1α expression. At greater arsenite concentrations, the release of the cation from the IP3R and the ensuing ERO1α expression remained unchanged but were nevertheless critical to sequentially promote concentration-dependent increases in Ca2+ release from the RyR, NADPH oxidase activation and a third mechanism of ERO1α expression which, in analogy to the one driven by mitochondrial superoxide, was also mediated by reactive oxygen species (ROS) and devoid of effects on Ca2+ homeostasis. Thus, concentration-independent stimulation of Ca2+ release from the IP3R is of pivotal importance for the effects of arsenite on Ca2+ homeostasis. It stimulates the expression of a fraction of ERO1α that primes the RyR to respond to the metalloid with concentration-dependent Ca2+-release, triggering the formation of superoxide in the mitochondrial respiratory chain and via NADPH oxidase activation. The resulting dose-dependent ROS formation was associated with a progressive increase in ERO1α expression, which however failed to affect Ca2+ homeostasis, thereby suggesting that ROS, unlike IP3R-dependent Ca2+ release, promote ERO1α expression in sites distal from the RyR.Keywords:
Homeostasis
Mitochondrial ROS
Interferon-gamma (IFN-gamma) had been shown to increase superoxide production of cultured monocytes from patients with chronic granulomatous disease (CGD). To elucidate the mechanism of the IFN-gamma-induced improvement of superoxide production of cultured monocytes from patients with CGD we examined the influence of IFN-gamma on the expression and the activity of the NADPH-oxidase in the monocytic cell-line Mono Mac 6. After cultivation of Mono Mac 6-cells in the presence of 500 U/ml IFN-gamma the superoxide production was found to be increased as well as the expression of the p47-phox cytosolic protein of the phagocytic NADPH-oxidase.
Chronic Granulomatous Disease
Phagocyte
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Left ventricular hypertrophy (LVH) is an independent marker of mortality in hypertension. Although the mechanisms contributing to LVH are complex, inflammation and oxidative stress may favor its development. We analyzed the association of the phagocytic NADPH oxidase–mediated superoxide anion release and LVH in patients with essential hypertension and the role of cardiotrophin-1 (CT-1) and interleukin-6 (IL-6), cytokines implicated in cardiac growth. Blood pressure, echocardiography data, and serum CT-1 and IL-6 levels were obtained in 140 subjects: 18 normotensives without LVH, 42 hypertensives without LVH, and 80 hypertensives with LVH. The NADPH oxidase–dependent superoxide production was assessed by chemiluminescence in peripheral blood mononuclear cells. Peripheral blood mononuclear cells were stimulated with CT-1 in vitro. Superoxide anion production by peripheral blood mononuclear cells associated with LVH and correlated with the left ventricular mass index. Serum CT-1 and IL-6 levels, which associated with the left ventricular mass index, correlated with superoxide production. Serum CT-1 and IL-6 levels were correlated. CT-1 stimulated NADPH oxidase superoxide production in peripheral blood mononuclear cells, which resulted in an increased release of IL-6. Our results show that superoxide anion production by the phagocytic NADPH oxidase associates with hypertensive heart disease, being significantly enhanced in hypertensive patients with LVH. This may be attributable to the activation of the NADPH oxidase by CT-1 and the subsequent release of IL-6. The phagocytic NADPH oxidase may be a therapeutic target in hypertensive heart disease.
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To identify the presence of endogenous superoxide anion-generating system in rabbit lens epithelial cells.The generation of superoxide anion was quantified by using lucigenin-amplified chemiluminescence (LUCL) in intact rabbit lens epithelial cells (N/N 1003A). The LUCL readings were recorded with a luminometer (LumiStar BMG) immediately upon arachidonic acid (AA) addition. To test the effects of superoxide dismutase (SOD) and DPI (non-specific inhibitor to NADPH oxidase), cells were preincubated for 30 minutes with indicated concentrations of inhibitors before stimulation with AA. The expression of NOX family, including NOX1, NOX2 or gp91(phox), NOX3, NOX4 and NOX5, was detected by RT-PCR.AA at dosage of 30-90 microM proportionally induced luminescence in N/N 1003A cells. Production of superoxide occurred quickly within 30 seconds of AA addition, reached the highest level after 200-260 seconds and dissipated after 600 seconds. The amount of superoxide anions, expressed as relative light unit of luminescence (RLU), was proportional to the concentration of AA used. Dose-dependent effect could be seen. Superoxide generation was inhibited in N/N1003A cells preloaded with SOD. DPI eliminated AA-induced superoxide anion generation. RT-PCR using primers specific for mRNAs of the five isoforms of the NOX proteins documented that mRNA encoding NOX1 through NOX5 were constitutively present in N/N1003A cells. The expression of NOX1 was much weaker than that of the other four NOX isoforms.NADPH oxidase complex is involved in superoxide anion generation in rabbit lens epithelial cells. N/N 1003A cells constitutively produce mRNA encoding five isoforms of NOX proteins.
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Abstract The Nef protein of the human immunodeficiency virus type 1 (HIV‐1) plays a crucial role in AIDS pathogenesis by modifying host cell signaling pathways. We investigated the effects of Nef on the NADPH oxidase complex, a key enzyme involved in the generation of reactive oxygen species during the respiratory burst in human monocyte/macrophages. We have recently shown that the inducible expression of HIV‐1 Nef in human macrophages cell line modulates in bi‐phasic mode the superoxide anion release by NADPH oxidase, inducing a fast increase of the superoxide production, followed by a delayed strong inhibition mediated by Nef‐induced soluble factor(s). Our study is focused on the molecular mechanisms involved in Nef‐mediated activation of NADPH oxidase and superoxide anion release. Using U937 cells stably transfected with different Nef alleles, we found that both Nef membrane localization and intact SH3‐binding domain are needed to induce superoxide release. The lack of effect during treatment with a specific MAPK pathway inhibitor, PD98059, demonstrated that Nef‐induced superoxide release is independent of Erk1/2 phosphorylation. Furthermore, Nef induced the phosphorylation and then the translocation of the cytosolic subunit of NADPH oxidase complex p47 phox to the plasma membrane. Adding the inhibitor PP2 prevented this process, evidencing the involvement of the Src family kinases on Nef‐mediated NADPH oxidase activation. In addition, LY294002, a specific inhibitor of phosphoinositide 3‐kinase (PI3K) inhibited both the Nef‐induced p47 phox phosphorylation and the superoxide anion release. These data indicate that Nef regulates the NADPH oxidase activity through the activation of the Src kinases and PI3K. J. Cell. Biochem. 106: 812–822, 2009. © 2009 Wiley‐Liss, Inc.
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Elevation of blood homocysteine (Hcy) levels (hyperhomocysteinemia) is a risk factor for cardiovascular disorders. We previously reported that oxidative stress contributed to Hcy-induced inflammatory response in vascular cells. In this study, we investigated whether NADPH oxidase was involved in Hcy-induced superoxide anion accumulation in the aorta, which leads to endothelial dysfunction during hyperhomocysteinemia. Hyperhomocysteinemia was induced in rats fed a high-methionine diet. NADPH oxidase activity and the levels of superoxide and peroxynitrite were markedly increased in aortas isolated from hyperhomocysteinemic rats. Expression of the NADPH oxidase subunit p22 phox increased significantly in these aortas. Administration of an NADPH oxidase inhibitor (apocynin) not only attenuated aortic superoxide and peroxynitrite to control levels but also restored endothelium-dependent relaxation in the aortas of hyperhomocysteinemic rats. Transfection of human endothelial cells or vascular smooth muscle cells with p22 phox siRNA to inhibit NADPH oxidase activation effectively abolished Hcy-induced superoxide anion production, thus indicating the direct involvement of NADPH oxidase in elevated superoxide generation in vascular cells. Taken together, these results suggest that Hcy-stimulated superoxide anion production in the vascular wall is mediated through the activation of NADPH oxidase, which leads to endothelial dysfunction during hyperhomocysteinemia.
Apocynin
Hyperhomocysteinemia
NOX1
Endothelial Dysfunction
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Granulocytes generate a "respiratory burst" of NADPH oxidase-dependent superoxide anion (O(2)(-*)) production that is required for efficient clearance of bacterial pathogens. Hv1 mediates a voltage-gated H(+) channel activity that is proposed to serve a charge-balancing role in granulocytic phagocytes such as neutrophils and eosinophils. Using mice in which the gene encoding Hv1 is replaced by beta-Geo reporter protein sequence, we show that Hv1 expression is required for measurable voltage-gated H(+) current in unstimulated phagocytes. O(2)(-*) production is substantially reduced in the absence of Hv1, suggesting that Hv1 contributes a majority of the charge compensation required for optimal NADPH oxidase activity. Despite significant reduction in superoxide production, Hv1(-/-) mice are able to clear several types of bacterial infections.
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Chronic Granulomatous Disease
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Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233–240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20–100 μM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCα, PKCβ and PKCγ) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCβ oligonucleotide, but not antisense PKCα oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCβ inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.
Apocynin
Staurosporine
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Reactive oxygen species elicit vascular effects ranging from acute dilatation because of hydrogen peroxide-mediated opening of K(+) channels to contraction arising from superoxide-dependent inactivation of endothelium-derived nitric oxide. Given that NADPH oxidases are major sources of superoxide in the vascular wall, this study examined the effects of exogenous NADPH, a substrate of these enzymes, on superoxide generation and isometric tone in mouse isolated aortic rings. NADPH caused concentration-dependent increases in superoxide generation (measured by lucigenin-enhanced chemiluminescence) and vascular tone (isometric tension recordings). However, surprisingly, whereas oxidized NADP(+) was unable to support superoxide production, it was equally as effective as reduced NADPH at stimulating vasocontraction. In addition, an NADPH oxidase inhibitor, diphenyleneiodonium, markedly attenuated NADPH-induced superoxide production, yet had no effect on vasocontractions to NADPH. In contrast, a broad specificity P2X receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, as well as the P2X1 selective antagonist, NF023, markedly attenuated both endothelium-dependent and -independent vasocontractions to NADPH, as did the P2X-desensitizing agent alpha,beta-methylene-ATP. Importantly, alpha,beta-methylene-ATP had no effect on superoxide production induced by NADPH. In conclusion, these findings suggest little role for NADPH oxidase-derived superoxide in the contractile effects of NADPH in the mouse aorta. Rather, NADPH seems to act as an agonist at two distinct P2X receptor populations; one located on the endothelium and the other on smooth muscle layer, both of which ultimately lead to contraction.
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Monocyte extravasation into the vessel wall has been shown to be a critical step in the development of atherosclerosis. Upon activation, monocytes produce a burst of superoxide anion due to activation of the NADPH oxidase enzyme complex. Monocyte-derived superoxide anion contributes to oxidant stress in inflammatory sites, is required for monocyte-mediated LDL oxidation, and alters basic cell functions such as adhesion and proliferation. We hypothesize that monocyte-derived superoxide anion production contributes to atherosclerotic lesion formation. In this brief review, we summarize our current understanding of the signal transduction pathways regulating NADPH oxidase activation and related superoxide anion production in activated human monocytes. Novel pathways are identified that may serve as future targets for therapeutic intervention in this pathogenic process. The contributions of superoxide anion and NADPH oxidase to atherogenesis are discussed. Future experiments are needed to clarify the exact role of NADPH oxidase-derived superoxide anion in atherogenesis, particularly that derived from monocytes.
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Respiratory burst
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