Identification of a superoxide-generating NADPH oxidase system in human fibroblasts.
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ABSTRACT:
SUBMITTER: Meier B
PROVIDER: S-EPMC1150038 | biostudies-other | 1991 Apr
REPOSITORIES: biostudies-other
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Project description:The membrane potential of cytoplasts, derived from human neutrophils, was depolarized by the activation of the superoxide-generating NADPH-dependent oxidase. The extent of the depolarization was inhibited by diphenylene iodonium and was therefore due directly to the activity of the oxidase, which must be electrogenic. The extent of the depolarization was influenced by alteration of the delta pH across the cytoplast membrane, indicating that the outward translocation of H+ eventually compensates for superoxide generation. The depolarization of the potential is enhanced by Cd2+, a blocker of H+ currents, suggesting that the compensatory movement is via an H+ channel.
| S-EPMC1148280 | biostudies-other
Project description:Superoxide (O(2) (.-)) may function as a second messenger or regulator of signal transduction when produced at low concentrations in the proper locations within cells. The purpose of these studies was to determine whether human corneal stromal (HCS) fibroblasts are capable of producing O(2) (.-) via nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, a family of protein complexes believed to be responsible for the localized and limited production of O(2) (.-) with regulatory activity.HCS cells, grown as primary and low-passage cultures of fibroblasts, were used as the sources of RNA for reverse transcriptase PCR, with primers specific for mRNAs encoding the proteins that comprise NADPH oxidases. Small interfering (si)RNAs were used to knockdown specific NOX mRNAs. Proteins composing the NADPH oxidase complexes were identified using western blots. The production of O(2) (.-) by whole cells and cell-free preparations was assessed by measurement of NADPH-dependent superoxide dismutase-inhibitable cytochrome c reduction.Whole cells and cell-free extracts of corneal stromal fibroblasts produced O(2) (.-) in an NADPH-dependent manner. These fibroblasts constitutively produced mRNAs encoding eight proteins known to comprise NADPH oxidase complexes. mRNAs encoding NOX1, NOX4, NOX5, p22 phox, p47 phox, p67 phox, and p40 phox as well as Rac were expressed. Treatment of HCS fibroblasts with siRNA pools specific for each of these three NOXs significantly reduced the steady state levels of the respective mRNAs. Western blots confirmed the existence of all the proteins required for O(2) (.-) production. Rac 1, a regulator of the activity of some forms of NADPH complexes was present in membranous cell fractions containing the oxidase proteins.HCS fibroblasts produced O(2) (.-) in a NADPH-dependent manner via at least three isoforms of NADPH oxidase. These cells expressed NOX1, NOX4, NOX5, p22 phox, p47 phox, p67 phox, and p40 phox as well as Rac. SiRNAs directed against each of the three putative isoforms of NOX significantly reduced the steady state levels of the appropriate NOX mRNA pools, thus confirming the existence of the three isoforms. The O(2) (.-) produced by the NADPH oxidases in HCS fibroblasts is a potential contributor to signal transduction pathways and a regulator of gene expression as well as a potential participant in processes that occur during inflammation.
| S-EPMC2788617 | biostudies-literature
Project description:ObjectiveHyperglycemia has been recognized for decades to be an exacerbating factor in ischemic stroke, but the mechanism of this effect remains unresolved. Here, we evaluated superoxide production by neuronal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a possible link between glucose metabolism and neuronal death in ischemia-reperfusion.MethodsSuperoxide production was measured by the ethidium method in cultured neurons treated with oxygen-glucose deprivation and in mice treated with forebrain ischemia-reperfusion. The role of NADPH oxidase was examined using genetic disruption of its p47(phox) subunit and with the pharmacological inhibitor apocynin.ResultsIn neuron cultures, postischemic superoxide production and cell death were completely prevented by removing glucose from the medium, by inactivating NADPH oxidase, or by inhibiting the hexose monophosphate shunt that generates NADPH from glucose. In murine stroke, neuronal superoxide production and death were decreased by the glucose antimetabolite 2-deoxyglucose and increased by high blood glucose concentrations. Inactivating NADPH oxidase with either apocynin or deletion of the p47(phox) subunit blocked neuronal superoxide production and negated the deleterious effects of hyperglycemia.InterpretationThese findings identify glucose as the requisite electron donor for reperfusion-induced neuronal superoxide production and establish a previously unrecognized mechanism by which hyperglycemia can exacerbate ischemic brain injury.
| S-EPMC4304737 | biostudies-literature
Project description:Superoxide radical plays an important role in redox cell signaling and physiological processes; however, overproduction of superoxide or insufficient activity of antioxidants leads to oxidative stress and contributes to the development of pathological conditions such as endothelial dysfunction and hypertension. Meanwhile, the studies of superoxide in biological systems represent unique challenges associated with short lifetime of superoxide, insufficient reactivity of the superoxide probes, and lack of site-specific detection of superoxide. In this work we have developed 15N-and deuterium-enriched spin probe 15N-CAT1H for high sensitivity and site-specific detection of extracellular superoxide. We have tested simultaneous tracking of extracellular superoxide by 15N-CAT1H and intramitochondrial superoxide by conventional 14N-containing spin probe mitoTEMPO-H in immune cells isolated from spleen, splenocytes, under basal conditions or stimulated with inflammatory cytokines IL-17A and TNFα, NADPH oxidase activator PMA, or treated with inhibitors of mitochondrial complex I rotenone or complex III antimycin A. 15N-CAT1H provides two-fold increase in sensitivity and improves detection since EPR spectrum of 15N-CAT1 nitroxide does not overlap with biological radicals. Furthermore, concurrent use of cell impermeable 15N-CAT1H and mitochondria-targeted 14N-mitoTEMPO-H allows simultaneous detection of extracellular and mitochondrial superoxide. Analysis of IL-17A- and TNFα-induced superoxide showed parallel increase in 15N-CAT1 and 14N-mitoTEMPO signals suggesting coupling between phagocytic NADPH oxidase and mitochondria. The interplay between mitochondrial superoxide production and activity of phagocytic NADPH oxidase was further investigated in splenocytes isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice. Angiotensin II infusion in wild-type mice increased the extracellular basal splenocyte superoxide which was further enhanced by complex III inhibitor antimycin A, mitochondrial uncoupling agent CCCP and NADPH oxidase activator PMA. Nox2 depletion attenuated angiotensin II mediated stimulation and inhibited both extracellular and mitochondrial PMA-induced superoxide production. These data indicate that splenocytes isolated from hypertensive angiotensin II-infused mice are "primed" for enhanced superoxide production from both phagocytic NADPH oxidase and mitochondria. Our data demonstrate that novel 15N-CAT1H provides high sensitivity superoxide measurements and combination with mitoTEMPO-H allows independent and simultaneous detection of extracellular and mitochondrial superoxide. We suggest that this new approach can be used to study the site-specific superoxide production and analysis of important sources of oxidative stress in cardiovascular conditions.
| S-EPMC9366351 | biostudies-literature
Project description:Blood flow assessment employing Doppler techniques is a useful procedure in pregnancy evaluation, as it may predict pregnancy disorders coursing with increased uterine vascular impedance, as pre-eclampsia. While the local causes are unknown, emphasis has been put on reactive oxygen species (ROS) excessive production. As NADPH oxidase (NOX) is a ROS generator, it is hypothesized that combining Doppler assessment with NOX activity might provide useful knowledge on placental bed disorders underlying mechanisms. A prospective longitudinal study was performed in 19 normal course, singleton pregnancies. Fetal aortic isthmus (AoI) and maternal uterine arteries (UtA) pulsatility index (PI) were recorded at two time points: 20-22 and 40-41 weeks, just before elective Cesarean section. In addition, placenta and placental bed biopsies were performed immediately after fetal extraction. NOX activity was evaluated using a dihydroethidium-based fluorescence method and associations to PI values were studied with Spearman correlations. A clustering of pregnancies coursing with higher and lower PI values was shown, which correlated strongly with placental bed NOX activity, but less consistently with placental tissue. The study provides evidence favoring that placental bed NOX activity parallels UtA PI enhancement and suggests that an excess in oxidation underlies the development of pregnancy disorders coursing with enhanced UtA impedance.
| S-EPMC4412968 | biostudies-literature
Project description: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.
| S-EPMC8387964 | biostudies-literature
Project description:An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence.
| S-EPMC1144305 | biostudies-other
Project description:We previously showed that luminal flow stimulates thick ascending limb (TAL) superoxide (O(2)(-)) production by stretching epithelial cells and increasing NaCl transport, and reported that the major source of flow-induced O(2)(-) is NADPH oxidase (Nox). However, the specific Nox isoform involved is unknown. Of the three isoforms expressed in the kidney-Nox1, Nox2, and Nox4-we hypothesized that Nox4 is responsible for flow-induced O(2)(-) production in TALs. Measurable flow-induced O(2)(-) production at physiological flow rates of 0, 5, 10, and 20 nl/min was 5 ± 1, 9 ± 2, 36 ± 6, and 66 ± 8 AU/s, respectively. RT-PCR detected mRNA for all three Nox isoforms in the TAL. The order of RNA abundance was Nox2 > Nox4 >>> Nox1. Since all three isoforms are expressed in TALs and pharmacological inhibitors are not selective, we used rats transduced with siRNA and knockout mice. Nox4 siRNA knocked down Nox4 mRNA expression by 63 ± 7% but did not reduce Nox1 or Nox2 mRNA. Flow-induced O(2)(-) was 18 ± 9 AU/s in TALs transduced with Nox4 siRNA compared with 77 ± 9 AU/s in tubules transduced with scrambled siRNA. Flow-induced O(2)(-) was 81 ± 5 AU/s in Nox2 knockout mice compared with 83 ± 13 AU/s in wild-type mice. In TALs transduced with Nox1 siRNA, flow-induced O(2)(-) was 82 ± 7 AU/s. We conclude that Nox4 mediates flow-induced O(2)(-) production in TALs.
| S-EPMC3469675 | biostudies-literature
Project description:Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development.
| S-EPMC4699827 | biostudies-literature
Project description:Nitric oxide (NO) is a ubiquitous cell signaling molecule which mediates widespread and diverse processes in the cell. These NO dependent effects often involve activation (e.g. NO binding to the heme group of soluble guanylyl cyclase for cGMP production) or inactivation (e.g. S-nitrosation) of protein targets. We studied the effect of NO and heme-NO on the transmembrane signaling enzyme NADPH oxidase 5 (NOX5), a heme protein which produces superoxide in response to increases in intracellular calcium. We found that treatment with NO donors increases NOX5 activity through heme-dependent effects, and that this effect could be recapitulated by the addition of heme-NO. This work adds to our understanding of NOX5 regulation in the cell but also provides a framework for understanding how NO could cause widespread changes in hemeprotein activity based on different affinities for heme v. heme-NO, and helps explain the opposing roles NO plays in activation and inactivation of hemeprotein targets.
| S-EPMC8355125 | biostudies-literature