Fetal-maternal interface impedance parallels local NADPH oxidase related superoxide production.
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ABSTRACT:
SUBMITTER: Guedes-Martins L
PROVIDER: S-EPMC4412968 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
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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.
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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.
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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.
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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.
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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.
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Project description:Coxsackievirus B infections are suspected environmental triggers of type 1 diabetes (T1D) and macrophage antiviral responses may provide a link to virus-induced T1D. We previously demonstrated an important role for NADPH oxidase (NOX)-derived superoxide production during T1D pathogenesis, as NOX-deficient NOD mice (NOD.Ncf1m1J ) were protected against T1D due, in part, to impaired proinflammatory TLR signaling in NOD.Ncf1m1J macrophages. Therefore, we hypothesized that loss of NOX-derived superoxide would dampen diabetogenic antiviral macrophage responses and protect from virus-induced diabetes. Upon infection with a suspected diabetogenic virus, Coxsackievirus B3 (CB3), NOD.Ncf1m1J mice remained resistant to virus-induced autoimmune diabetes. A concomitant decrease in circulating inflammatory chemokines, blunted antiviral gene signature within the pancreas, and reduced proinflammatory M1 macrophage responses were observed. Importantly, exogenous superoxide addition to CB3-infected NOD.Ncf1m1J bone marrow-derived macrophages rescued the inflammatory antiviral M1 macrophage response, revealing reduction-oxidation-dependent mechanisms of signal transducer and activator of transcription 1 signaling and dsRNA viral sensors in macrophages. We report that superoxide production following CB3 infection may exacerbate pancreatic β cell destruction in T1D by influencing proinflammatory M1 macrophage responses, and mechanistically linking oxidative stress, inflammation, and diabetogenic virus infections.
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Project description:Key pointsLRRC8A-containing anion channels associate with NADPH oxidase 1 (Nox1) and regulate superoxide production and tumour necrosis factor-α (TNFα) signalling. Here we show that LRRC8C and 8D also co-immunoprecipitate with Nox1 in vascular smooth muscle cells. LRRC8C knockdown inhibited TNFα-induced O2 •- production, receptor endocytosis, nuclear factor-κB (NF-κB) activation and proliferation while LRRC8D knockdown enhanced NF-κB activation. Significant changes in LRRC8 isoform expression in human atherosclerosis and psoriasis suggest compensation for increased inflammation. The oxidant chloramine-T (ChlorT, 1 mM) weakly (∼25%) inhibited LRRC8C currents but potently (∼80%) inhibited LRRC8D currents. Substitution of the extracellular loop (EL1, EL2) domains of 8D into 8C conferred significantly stronger (69%) ChlorT-dependent inhibition. ChlorT exposure impaired subsequent current block by DCPIB, which occurs through interaction with EL1, further implicating external oxidation sites. LRRC8A/C channels most effectively sustain Nox1 activity at the plasma membrane. This may result from their ability to remain active in an oxidized microenvironment.AbstractTumour necrosis factor-α (TNFα) activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs), producing superoxide (O2 •- ) required for subsequent signalling. LRRC8 family proteins A-E comprise volume-regulated anion channels (VRACs). The required subunit LRRC8A physically associates with Nox1, and VRAC activity is required for Nox activity and the inflammatory response to TNFα. VRAC currents are modulated by oxidants, suggesting that channel oxidant sensitivity and proximity to Nox1 may play a physiologically relevant role. In VSMCs, LRRC8C knockdown (siRNA) recapitulated the effects of siLRRC8A, inhibiting TNFα-induced extracellular and endosomal O2 •- production, receptor endocytosis, nuclear factor-κB (NF-κB) activation and proliferation. In contrast, siLRRC8D potentiated NF-κB activation. Nox1 co-immunoprecipitated with 8C and 8D, and colocalized with 8D at the plasma membrane and in vesicles. We compared VRAC currents mediated by homomeric and heteromeric LRRC8C and LRRC8D channels expressed in HEK293 cells. The oxidant chloramine T (ChlorT, 1 mM) weakly inhibited 8C, but potently inhibited 8D currents. ChlorT exposure also impaired subsequent current block by the VRAC blocker DCPIB, implicating external sites of oxidation. Substitution of the 8D extracellular loop domains (EL1, EL2) into 8C conferred significantly stronger ChlorT-mediated inhibition of 8C currents. Our results suggest that LRRC8A/C channel activity can be effectively maintained in the oxidized microenvironment expected to result from Nox1 activation at the plasma membrane. Increased ratios of 8D:8C expression may potentially depress inflammatory responses to TNFα. LRRC8A/C channel downregulation represents a novel strategy to reduce TNFα-induced inflammation.
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Project description:Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO2) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO2 to regulate the production of reactive oxygen species under proinflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O2(•-)) due to the NADPH-dependent univalent reduction of oxygen to O2(•-) by the phagocytic NADPH oxidase isoform (NOX2), the activity of NOX2 being the main source of O2(•-) in monocytes/macrophages. Because the NOX2 and AA pathways are connected, we propose that AANO2 can modulate macrophage activation by inhibiting O2(•-) formation by NOX2. When macrophages were activated in the presence of AANO2, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex red fluorescence, and flow cytometry; this process also occurs under physiological mimic conditions within the phagosomes. AANO2 decreased O2(•-) production in a dose- (IC50=4.1±1.8 μM AANO2) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g., p40(phox) and p47(phox)), as analyzed by immunoprecipitation and Western blot. However, a reduction in the migration to the membrane of p47(phox) was obtained, suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, in which subcutaneous injection of AANO2 was able to decrease NOX2 activity in macrophages from thioglycolate-treated mice.
| S-EPMC3622795 | biostudies-literature
Project description:Redox-regulated signal transduction is coordinated by spatially controlled production of reactive oxygen species within subcellular compartments. The nucleus has long been known to produce superoxide (O(2)(·-)); however, the mechanisms that control this function remain largely unknown. We have characterized molecular features of a nuclear superoxide-producing system in the mouse liver. Using electron paramagnetic resonance, we investigated whether several NADPH oxidases (NOX1, 2, and 4) and known activators of NOX (Rac1, Rac2, p22(phox), and p47(phox)) contribute to nuclear O(2)(·-) production in isolated hepatic nuclei. Our findings demonstrate that NOX4 most significantly contributes to hepatic nuclear O(2)(·-) production that utilizes NADPH as an electron donor. Although NOX4 protein immunolocalized to both nuclear membranes and intranuclear inclusions, fluorescent detection of NADPH-dependent nuclear O(2)(·-) predominantly localized to the perinuclear space. Interestingly, NADP(+) and G6P also induced nuclear O(2)(·-) production, suggesting that intranuclear glucose-6-phosphate dehydrogenase (G6PD) can control NOX4 activity through nuclear NADPH production. Using G6PD mutant mice and G6PD shRNA, we confirmed that reductions in nuclear G6PD enzyme decrease the ability of hepatic nuclei to generate O(2)(·-) in response to NADP(+) and G6P. NOX4 and G6PD protein were also observed in overlapping microdomains within the nucleus. These findings provide new insights on the metabolic pathways for substrate regulation of nuclear O(2)(·-) production by NOX4.
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