Measured Superoxide Production Research Articles

Demonstrating drug treatment efficacies by monitoring superoxide dynamics in human lung cancer cells with time-lapse fluorescence microscopy.

Metformin hydrochloride, an antihyperglycemic agent, and sulindac, a nonsteroidal anti-inflammatory drug, are FDA-approved drugs known to exert anticancer effects. Previous studies demonstrated sulindac and metformin's anticancer properties through mitochondrial dysfunction and inhibition of mitochondrial electron transport chain complex I and key signaling pathways. In this study, various drugs were administered to A549 lung cancer cells, and results revealed that a combination of sulindac and metformin enhanced cell death compared to the administration of the drugs separately. To measure superoxide production over time, we employed a time-lapse fluorescence imaging technique using mitochondrial-targeted hydroethidine. Fluorescence microscopy data showed the most significant increases in superoxide production in the combination treatment of metformin and sulindac. Results showed significant differences between the combined drug treatment and control groups and between the positive control and control groups. This approach can be utilized to quantify the anticancer efficacy of drugs, creating possibilities for additional therapeutic options.

Use of Electron Paramagnetic Resonance (EPR) to Evaluate Redox Status in a Preclinical Model of Acute Lung Injury.

Patients with hyper- vs. hypo-inflammatory subphenotypes of acute respiratory distress syndrome (ARDS) exhibit different clinical outcomes. Inflammation increases the production of reactive oxygen species (ROS) and increased ROS contributes to the severity of illness. Our long-term goal is to develop electron paramagnetic resonance (EPR) imaging of lungs in vivo to precisely measure superoxide production in ARDS in real time. As a first step, this requires the development of in vivo EPR methods for quantifying superoxide generation in the lung during injury, and testing if such superoxide measurements can differentiate between susceptible and protected mouse strains. In WT mice, mice lacking total body extracellular superoxide dismutase (EC-SOD) (KO), or mice overexpressing lung EC-SOD (Tg), lung injury was induced with intraperitoneal (IP) lipopolysaccharide (LPS) (10mg/kg). At 24h after LPS treatment, mice were injected with the cyclic hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) or 4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid (DCP-AM-H) probes to detect, respectively, cellular and mitochondrial ROS - specifically superoxide. Several probe delivery strategies were tested. Lung tissue was collected up to one hour after probe administration and assayed by EPR. As measured by X-band EPR, cellular and mitochondrial superoxide increased in the lungs of LPS-treated mice compared to control. Lung cellular superoxide was increased in EC-SOD KO mice and decreased in EC-SOD Tg mice compared to WT. We also validated an intratracheal (IT) delivery method, which enhanced the lung signal for both spin probes compared to IP administration. We have developed protocols for delivering EPR spin probes in vivo, allowing detection of cellular and mitochondrial superoxide in lung injury by EPR. Superoxide measurements by EPR could differentiate mice with and without lung injury, as well as mouse strains with different disease susceptibilities. We expect these protocols to capture real-time superoxide production and enable evaluation of lung EPR imaging as a potential clinical tool for subphenotyping ARDS patients based on redox status.

Abstract MP08: New Function Of Sox6 In Renovascular Hypertension Induced Oxidative Stress And Kidney Damage Through Renin, Prorenin And The (pro)renin Receptor Expression Control.

Introduction: During renovascular hypertension induced by renal artery stenosis (RAStenosis), smooth muscle cells are recruited to produce and release renin compensating for the decrease in renal perfusion. In renovascular hypertension, renin expression increases causing a surge in angiotensin II, oxidative stress and kidney damage. Here we present a new function of the transcription factor Sox6 in kidney injury and oxidative damage during RAStenosis. Methods: We used a mouse model in which Sox6 is knockout specifically in renin expressing cells (Ren1d Cre /Sox6 fl/fl -Sox6-KO). We developed a modified 2-kidney 1-clip (2K1C) Goldblatt model to induce RAStenosis. We measured superoxide production (pmol/mg pr) in kidney using high-performance liquid chromatography in Sox6-KO and Ren1d Cre /Sox6 wt/wt (Sox6-WT) littermates. Renin, prorenin, (pro)renin receptor (PRR) and N-GAL expressions were measured using Western blot using β-actin as housekeeping gene. Western blot band density analysis was performed using ImageJ. Kidney injury was determined by measuring creatinine clearance. Results: We found that Sox6-KO mice exhibit lower expression of renin (0.36 vs. 0.45, SEM= 0.11 n= 11-15, p<0.01), and prorenin (1.25 vs. 2.07, SEM=0.30, n=11-15, p<0.05) and mice are protected against renovascular hypertension (SBP, 114.5 vs. 131.1, SEM=5.31 n=13-15, p<0.01), and kidney injury measuring N-GAL expression (1.3 vs. 2.7, SEM=0.38, n=6-8, p<0.01) and creatinine clearance (2372 vs. 1341, SEM=342.3, n=4-5, p<0.05). Furthermore, we found that the levels of superoxide were significantly lower in Sox6-KO mice compared to Sox6-WT littermates (218 vs 47, SEM=50.6, N=7, P<0.01). The expression of PRR was significantly lower in Sox6-KO compared to Sox6-WT during RAStenosis (1.23 vs. 0.72, SEM=0.33, n=2-5, p<0.01). Conclusions: Our results show that Sox6 depletion in renin expressing cells inhibits the increase in renovascular hypertension and prevents the increase in renin, prorenin, PRR, and N-GAL expression as well as superoxide production, preserving creatinine clearance during RAStenosis. These results suggest that Sox6 has a new function in hypertension and kidney injury induced by RAS.

Measurement of Superoxide Production in Acute Hypoxia by Fixed-Cell Microscopy.

Redox signaling implication in cell adaptation to hypoxia has been studied for a long time, both in long-term and acute responses. However, measurement of superoxide and other reactive oxygen species (ROS) in acute hypoxia is technically challenging, for example, because of the need to overcome the effect of cell reoxygenation before measurement.Here we describe a method we have developed for measuring superoxide production in acute hypoxia using the fluorescent probe dihydroethidine in fixed-cell microscopy. The method allows measuring the kinetics of superoxide production (or other ROS with the appropriate probes) by incubating the probe in different time windows during hypoxia incubation.

Synthesis and characterization of a 5-membered ring cyclic hydroxylamine coupled to triphenylphosphonium to detect mitochondrial superoxide by EPR spectrometry

As mitochondrial superoxide is becoming an attractive metabolic and pharmacological target, there is an important need for developing analytical tools able to detect superoxide with high sensitivity and specificity. Among EPR-based methods, it has been recently reported that cyclic hydroxylamines offer a high sensitivity to measure superoxide production. Here, we report the synthesis and evaluation of mitoCPH, in which a 5-membered ring hydroxylamine was coupled to a triphenylphosphonium moiety to allow mitochondrial accumulation. MitoCPH efficiently reacted with superoxide with a bimolecular rate constant of 1.5 × 104 M−1 s−1. We assessed the ability of this compound to detect superoxide in PBS buffer, lysates, and in paraquat-stimulated cells. We compared its performance with CMH, a nontargeted 5-membered ring hydroxylamine, and mitoTEMPO-H, a classically used 6-membered ring hydroxylamine targeted to mitochondria. MitoCPH presented a higher sensitivity for superoxide anion detection than commonly used mitoTEMPO-H, both in buffer and in cell lysates. While we have described the ability of mitoCPH to detect superoxide in different cellular media, we cannot exclude other potential contributors to the nitroxide production from this probe. Therefore, mitoCPH should be considered as a mitochondria-targeted probe and its use as selective superoxide probe should be used cautiously.

Impairment of Mitochondrial Redox Status in Peripheral Lymphocytes of Multiple Sclerosis Patients.

Literature suggests that oxidative stress (OS) may be involved in the pathogenesis of multiple sclerosis (MS), in which the immune system is known to play a key role. However, to date, the OS in peripheral lymphocytes and its contribution to the disease remain unknown. The aim of the present study was to explore the influence of OS in peripheral lymphocytes of MS patients. To that end, a cross-sectional, observational pilot study was conducted [n = 58: 34 MS and 24 healthy subjects (control group)]. We have measured superoxide production and protein mitochondrial complex levels in peripheral blood mononuclear cells (PBMCs) isolated from MS patients and control. Lactate levels and the antioxidant capacity were determined in plasma. We adjusted the comparisons between study groups by age, sex and cell count according to case. Results demonstrated that PBMCs, specifically T cells, from MS patients exhibited significantly increased superoxide anion production compared to control group (p = 0.027 and p = 0.041, respectively). Increased superoxide production in PBMCs was maintained after the adjustment (p = 0.044). Regarding mitochondrial proteins, we observe a significant decrease in the representative protein content of the mitochondrial respiratory chain complexes I-V in PBMCs of MS patients (p = 0.002, p = 0.037, p = 0.03, p = 0.044, and p = 0.051, respectively), which was maintained for complexes I, III, and V after the adjustment (p = 0.026; p = 0.033; p = 0.033, respectively). In MS patients, a trend toward increased plasma lactate concentration was detected [8.04 mg lactate/dL (5.25, 9.49) in the control group, 11.36 mg lactate/dL (5.41, 14.81) in MS patients] that was statistically significant after the adjustment (p = 0.013). This might be indicative of compromised mitochondrial function. Finally, antioxidant capacity was also decreased in plasma from MS patients, both before (p = 0.027) and after adjusting for sex and age (p = 0.006). Our findings demonstrate that PBMCs of MS patients show impaired mitochondrial redox status and deficient antioxidant capacity. These results demonstrate for the first time the existence of mitochondrial alterations in the cells immune cells of MS patients already at the peripheral level.

Superoxide generation via the NR2B-NMDAR/RasGRF1/NOX2 pathway promotes dendritogenesis.

N-methyl-D-aspartate receptors (NMDARs) that contain the NR2A and NR2B subunits play a critical role in neuronal plasticity and dendritogenesis. Gain-and-loss-of function studies indicate that NR2B, but not NR2A, promotes dendritic branching. Accumulating evidence indicates that stimulation of NMDARs activates NADPH oxidase (NOX2), thereby generating superoxide. However, the molecular underpinnings of this process are not understood. RasGRF1, a guanine nucleotide exchange factor, is key for several forms of neuronal plasticity and interacts directly with the tail of NR2B. We investigated whether the NR2B-NMDAR/RasGRF1 pathway regulates the activity of NOX2 and whether superoxide production is required for dendritogenesis. We measured superoxide production in developing primary cultures of hippocampal neurons from 3 to 25 days in vitro (DIV) with the probe dihydroethidium (dHE). We found the highest dHE levels at early and intermediate developmental stages (3-15 DIV), when the NR2B-NMDAR expression is abundant. During these early/intermediate developmental stages, but not in mature neurons (>15 DIV), NMDAR activity is required for superoxide production. We also found that disrupting the NR2B-RasGRF1 interaction led to reduced dHE fluorescence intensity and moreover inhibited dendritic branching in hippocampal neurons. Together, our data indicate that superoxide production is induced by the NR2B-NMDARs/RasGRF1/NOX2 pathway and promotes dendritogenesis.

SAT-175 (-)-Epicatechin Modulates Cellular Signaling and Mitochondrial Redox in Endothelium

Diabetes mellitus affects 451 million people worldwide, and people with diabetes are 3-5 times more likely to develop cardiovascular disease. In vascular tissue, mitochondrial function is important for vasodilation and arterial stiffness, both of which are abnormal in diabetes. Diabetes-mediated generation of excess reactive oxygen species (ROS) may be contributing to this problem. We have identified (-)-epicatechin (EPICAT), a plant compound and known vasodilator, as a potential therapy. We hypothesized that mitochondrial ROS accumulation in cells treated with high glucose (HG) or antimycin A (AA, a compound targeting complex III) could be diminished by EPICAT. We further hypothesized that reduction of ROS would correlate with improved mitochondrial dynamics and cellular signaling. To test this hypothesis, we employed human umbilical vein endothelial cells (HUVEC) treated with HG, AA, and/or 0.1 or 1.0 µM of EPICAT, and measured mitochondrial and cellular superoxide, mitochondrial dynamics, and cellular signaling upstream of mitochondrial function. AA significantly increased mitochondrial width (p=0.012) and decreased mitochondrial length (p=0.0001), but we observed no impact of EPICAT. We measured superoxide production by electron paramagnetic resonance spectroscopy using two spin probes specific to mitochondrial (Mito-TEMPO-H) and total cell (CMH) superoxide. No impact of HG treatment was detected in the total cell superoxide; however, an increase in mitochondrial superoxide was measured in cells treated with HG as compared to control (0.42 µM superoxide vs. 0.31 µM superoxide, respectively). We found that both concentrations of EPICAT significantly attenuated mitochondrial superoxide production when treated with HG (HG alone=0.44 µM superoxide, HG + 0.1 µM EPICAT=0.34 µM superoxide, HG + 1.0 µM EPICAT= 0.34 µM superoxide, p=0.02 EPICAT effect), but not with AA treatment. We found that in combination with HG, 0.1 µM EPICAT significantly increased eNOS expression (p<0.05). At 1.0 µM, EPICAT also increased peNOS expression (p<0.05) and attenuated a decrease in pAMPK (p<0.05) in response to HG. EPICAT at both concentrations significantly increased mitochondrial complex II expression (p<0.05 for both). In summary, HG-induced mitochondrial ROS is responsive to EPICAT, whereas AA-induced ROS is not, suggesting that EPICAT is useful in mitigating moderate ROS concentrations and may be modulating mitochondrial activity upstream of complex III. Our data further illustrate that EPICAT may be a tool to modulate both cellular and mitochondrial glucose toxicity through the regulation of AMPK and eNOS, upstream of mitochondrial activity, as well as bolstering endogenous antioxidant defenses.

Acute Hyperbaric Oxygenation, Contrary to Intermittent Hyperbaric Oxygenation, Adversely Affects Vasorelaxation in Healthy Sprague-Dawley Rats due to Increased Oxidative Stress.

The present study was aimed at assessing endothelium-dependent vasorelaxation, at measuring superoxide production in the aorta and femoral artery, and at determining antioxidative enzyme expression and activity in aortas of male Sprague-Dawley rats (N = 135), randomized to an A-HBO2 group exposed to a single hyperbaric oxygenation session (120′ of 100% O2 at 2.0 bars), a 24H-HBO2 group (single session, examined 24 h after exposure), a 4D-HBO2 group (4 consecutive days of single sessions), and a CTRL group (untreated group). Vasorelaxation of aortic rings in response to acetylcholine (AChIR) and to reduced pO2 (HIR) was tested in vitro in the absence/presence of NOS inhibitor L-NAME and superoxide scavenger TEMPOL. eNOS, iNOS, antioxidative enzyme, and NADPH oxidase mRNA expression was assessed by qPCR. Serum oxidative stress markers and enzyme activity were assessed by spectrometry, and superoxide production was determined by DHE fluorescence. Impaired AChIR and HIR in the A-HBO2 group were restored by TEMPOL. L-NAME inhibited AChIR in all groups. Serum oxidative stress and superoxide production were increased in the A-HBO2 group compared to all other groups. The mRNA expression of iNOS was decreased in the A-HBO2 and 24H-HBO2 groups while SOD1 and 3 and NADPH oxidase were increased in the 4D-HBO2 group. The expression and activity of catalase and glutathione peroxidase were increased in the 4D-HBO2 group as well. AChIR was NO dependent. Acute HBO2 transiently impaired vasorelaxation due to increased oxidative stress. Vasorelaxation was restored and oxidative stress was normalized 24 h after the treatment.

78 Mito-TEMPO, a Scavenger for Mitochondria-Derived Reactive Oxygen Species, Enhances Porcine Pre-Implantation Embryo Development

The production of reactive oxygen species (ROS) from mitochondria contributes to redox signalling, mitochondrial functions, and apoptosis. However, the specific effects of mitochondria target superoxide (O2 •–) on porcine embryo development remain unclear. The objective of present study was to examine the differences of mitochondrial functions and dynamics in 2 subpopulations of porcine zygotes (G1 and G2), and to investigate the effects of Mito-TEMPO on porcine embryo development. Porcine embryos were visually classified in 2 groups [Grade (G)1: over 90%, and G2: below 90%] according to the lipid distribution at the zygote stage. The blastocyst development rate was greater in G1 than in G2 embryos (G1: 26.5 ± 5.9% v. G2: 16.2 ± 7.9%; P &lt; 0.05). To evaluate blastocyst quality, we performed a 4′,6-diamidino-2-phenylindole (DAPI)-TUNEL assay. The proportion of TUNEL-positive cells was higher (P &lt; 0.05) in G2 than G1 embryos. We measured superoxide production by MitoSOX staining as mitochondrial superoxide specific fluorescence dye by iRiSTM Digital Cell Image System (Logos Biosystems Inc., Gyeonggi-do, South Korea). Red fluorescence intensity of superoxide in G2 embryos significantly increased (P &lt; 0.05) compared with that in G1. We investigated changes in mitochondrial functions using a Mitotracker JC-1 mitochondrial membrane potential assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and ATP determination kit, respectively. Mitochondria membrane potential and ATP production were lower (P &lt; 0.05) in G2 embryos than in G1 embryos. To confirm the protein levels of mitochondria fission protein DRP1, we performed Western blot analysis (per 40 embryos). Phosphorylation DRP1-Ser616 was increased (P &lt; 0.05) in G1 embryos at cleavage stage compared with that in zygote, but not significantly different in G2 embryos. Thus, G2 embryos showed low development rate until blastocyst via mitochondrial dysfunction, increase in fission protein expression and mitochondrial aggregation according to the elevation of mito-ROS. Subsequently, the effect of the adding superoxide scavenger Mito-TEMPO was investigated in G2 embryos. Blastocyst formation (G2+MitoTempo: 28.8 ± 4.0% v. G2: 19.1 ± 5.1%; P &lt; 0.05) and mitochondrial aggregation were recovered by mito-ROS reduction mediated by Mito-TEMPO. Our observations demonstrated that regulation of superoxide in mitochondria is important in pre-implantation development of porcine embryos. This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01117604) and the Bio-industry Technology Development Program (316037-04-2-HD020) through the Rural Development Administration and the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.

Modulation of Heme Biosynthesis by Ferrochelatase Inhibition Controls Soluble Guanylate Cyclase Expression and Superoxide Production in Bovine Coronary Arteries

Nitric oxide interacts with the Fe2+ heme prosthetic group in soluble guanylate cyclase (sGC) stimulating the conversion of guanosine 5′‐triphosphate to cyclic guanosine 3′,5′‐monophosphate (cGMP). Adequate cGMP production is associated with protective mechanisms against multiple cardiovascular disease processes. Thus, disturbances in this signaling pathway can lead to many pathological consequences. Since there has been minimal investigation on the consequences of altering heme biosynthesis on the cardiovascular system, we investigated if mitochondrial heme biosynthesis is an important factor in controlling the expression and function of sGC and systems influencing superoxide generation and actions. For the purpose of this study, we induced a heme deficiency by inhibiting ferrochelatase (FECH) with N‐methyl protoporphyrin IX (NMPP), potent FECH inhibitor. Bovine coronary arteries (BCA) were treated for 24 hours with 10 μM NMPP. Chemiluminescence (5μM lucigenin) was used to measure superoxide production; western blot analysis was used to measure expression of FECH, sGC and other heme‐containing proteins; and a QuantiChrom heme assay kit was used to measure heme levels. NMPP treatment promoted sGC depletion and attenuation of its activity based on it decreasing protein kinase G‐mediated phosphorylation of VASP and attenuation of relaxation to a nitric oxide donor. NMPP also decreased the expression of heme proteins including cytochrome oxidase subunit 4 and catalase. In addition, superoxide production was significantly elevated in NMPP treated coronary arteries when compared to that in control samples, and scavenging extra mitochondrial superoxide with Tempol and mitochondrial matrix superoxide with MitoTempol attenuated the increase in superoxide. NMPP stimulated increases in superoxide were associated with a decrease expression of mitochondrial FECH expression. Thus, inhibition of FECH increased extra mitochondrial and mitochondrial superoxide and depleted sGC. Our studies suggest that disruption of heme biosynthesis resulting in a loss of cGMP production may serve as a contributing mechanism to the progression of cardiovascular disease.Support or Funding InformationFunded by NIH grants R01HL115124 &amp; R01HL129797

Measurement of Superoxide Production and NADPH Oxidase Activity by HPLC Analysis of Dihydroethidium Oxidation.

The fluorogenic probe dihydroethidium (DHE) is widely used for detecting intracellular superoxide. DHE oxidation by superoxide generates specifically the compound 2-hydroxyethidium (2-E+OH), so that 2-E+OH detection confers specificity to superoxide assessment among many other reactive oxygen species. However, DHE oxidation in biological systems leads to formation of other fluorescent products, particularly ethidium, usually formed at higher quantities than 2-E+OH. Since both 2-E+OH and ethidium are fluorescent, their identification and quantification is possible only after their physical separation by HPLC. Here we describe the detailed procedures for superoxide measurement in cells (adhered or not) and fresh tissues fragments, followed by acetonitrile extraction and simultaneous fluorescent detection of 2-E+OH and ethidium and absorbance detection of remaining unreacted DHE. In addition we report the use of DHE/HPLC for measuring NADPH oxidase activity in enriched-membrane fraction isolated from cells or tissues. These methods can improve accuracy and precision of quantitative superoxide measurements in biological samples.

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity.

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.

Abstract 123: A Fructose-enriched Diet Stimulates Superoxide Production in Juxtaglomerular Cells and Prevents High-salt Induced Inhibition of Plasma Renin Activity (PRA)

A fructose-enriched diet has been associated with hypertension. Western diets are rich in fructose and salt. We found that a fructose enriched diet plus high-salt induced salt sensitive hypertension. Plasma renin activity (PRA) is essential for blood pressure (BP) control. A high-salt diet decreases PRA by inhibiting renin release from juxtaglomerular (JG) cells. However it is not known if dietary fructose might impair the inhibition of renin release by high salt to promote salt sensitivity. Salt sensitive rats have enhanced levels of superoxide in the renal cortex, and we found that superoxide stimulates renin release from JG cells. Thus, we hypothesized that a fructose-enriched diet (20%) promotes salt sensitive hypertension in part by preventing high salt-induced inhibition of renin release from JG cells by enhancing superoxide production. To test this, Sprague Dawley rats were given 20% fructose in their drinking water, with normal or high salt diet (4% NaCl) for up to 4 weeks. Feeding normal rats a fructose+High-salt diet increased systolic BP by 30 mmHg whereas fructose or high-salt alone did not change BP (High-salt = 125±4, Fruct = 131±4, Fruct+High-salt = 147±7; n=6, p &lt;0.05). A high-salt diet alone for 4 weeks decreased PRA by 85%. However, in rats fed fructose+High-salt did not decrease PRA (in ng AII/ml/hr: Ctrl = 2.51±0.72, High-salt = 0.43±0.07, Fruct = 2.71±0.9, Fruct+High-salt = 1.89±0.43; n=10, p &lt;0.05). We next examined the role of the fructose or fructose+High-salt diet on NADPH oxidase expression in isolated JG cells. NOX4 expression was enhanced in JG cells from rats fed fructose+High-salt diet (n=4; p &lt;0.05). Next, we measured superoxide production with Dihydroethidium and found it was higher in JG cells from rats fed fructose+high-salt diet compared to high-salt alone (% of Ctrl: High-salt = 90.7±37; fruct+High-salt = 289±85; n=4; p &lt;0.05). We conclude that a 20% fructose-diet promotes salt sensitivity of BP. The mechanism may involve enhanced NOX4 expression and elevated superoxide levels within JG cells stimulating renin release. 15 million Americans consume 20% of their calories from fructose, and most, 4-8 times the recommended salt intake. Decreasing fructose intake could have a beneficial BP effect in hypertensive patients.

Nitro-Triarylmethyl Radical as Dual Oxygen and Superoxide Probe.

Superoxide radical is involved in numerous physiological and pathophysiological processes. Tetrathiatriarylmethyl (TAM) radicals are known to react with superoxide allowing measurement of superoxide production in biological media. We report the synthesis of a Nitro conjugated TAM radical showing a rate constant of 7×105M-1s-1 which is two order of magnitude higher than other TAMs, allowing high sensitivity measurement of superoxide.

Catherine Pasquier Memorial Award Lecture (shared): The adventure of detecting subtle signals (such as protein nitrosothiols and superoxide) among cell traffic jams (from proteomics to hypoxia)

Like big human cities, cells are crowded environments with a lot of inhabitants, where a myriad of signals try to orchestrate how each component can do its best among the crowd, and how the whole system respond to changes in the environment. Some signals are robust and stable, so they can be easily studied by life science researchers. However, free radical researchers know well that other molecular signals are short-lived and unstable. I have been involved in several studies that can be seen as adventures searching for those subtle signals, overcoming the difficulties that appear in the way. One is the reliable proteomic identification of protein S-nitrosylation (or S-nitrosation), and of other reversible oxidations of cysteines, in physiological scenarios where they can act as signals (not just in nitrosative, oxidative or similar stresses). Different techniques have been used for that purpose (called “thiol redox proteomics”), with technical developments still ongoing. A related adventure is the study of the functional relevance of these modifications in individual proteins, and of the particularities of these modes of signalling. Another adventure deals with the role of reactive oxygen species production in hypoxia sensing and adaptation, on which a debate has been maintained for a long time. We have measured superoxide production in acute hypoxia, showing there is a fast burst at the first minutes of acute hypoxia, and we are currently studying its molecular mechanism, implying mitochondrial complex I and the sodium/calcium exchanger. This can be a common signal to different cell types that produces diverse effects depending on the signalling cascades and effectors of each cell type. Thiol redox proteomics can help in identifying the specific protein cysteine residues that can be affected by reversible oxidation and take part in those signals.

Response to Comment on Sharma. Mitochondrial Hormesis and Diabetic Complications. Diabetes 2015;64:663–672

I appreciate the comments of Kumar et al. (1) on my recent article (2). Although I have followed the work and insights of Michael Brownlee (3), much to my surprise our group did not find any evidence of increased mitochondrial electron transport chain (ETC) activity and superoxide production in in vivo models of diabetic kidney disease (4). To my knowledge, our group's studies are the first in vivo imaging approach to measure superoxide production in diabetic kidneys, and there was no evidence of increased mitochondrial superoxide production in the diabetic kidney using different …

TCR-triggered extracellular superoxide production is not required for T-cell activation.

BackgroundIn the last decade, reactive oxygen species (ROS) production has been shown to occur upon T-cell receptor (TCR) stimulation and to affect TCR-mediated signalling. However, the exact reactive species that are produced, how ROS are generated and their requirement for T-cell activation, proliferation or cytokine production remain unclear, especially in the case of primary human T cells. Moreover, several groups have questioned that ROS are produced upon TCR stimulation.ResultsTo shed some light onto this issue, we specifically measured superoxide production upon TCR ligation in primary human and mouse T lymphocytes. We showed that superoxide is indeed produced and released into the extracellular space. Antioxidants, such as superoxide dismutase and ascorbate, abolished superoxide production, but surprisingly did not affect activation, proliferation and cytokine secretion in TCR-stimulated primary human T cells. It has been suggested that T cells produce ROS via the NADPH oxidase 2 (NOX2). Therefore, we investigated whether T-cell activation is affected in NOX2-deficient mice (gp91phox −/−). We found that T cells from these mice completely lack inducible superoxide production but display normal upregulation of activation markers and proliferation.ConclusionsCollectively, our data indicate that primary T cells produce extracellular superoxide upon TCR triggering, potentially via NOX2 at the plasma membrane. However, superoxide is not required for T-cell activation, proliferation and cytokine production.

Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

TCR-triggered extracellular superoxide production is not required for T-cell activation

BackgroundIn the last decade, reactive oxygen species (ROS) production has been shown to occur upon T-cell receptor (TCR) stimulation and to affect TCR-mediated signalling. However, the exact reactive species that are produced, how ROS are generated and their requirement for T-cell activation, proliferation or cytokine production remain unclear, especially in the case of primary human T cells. Moreover, several groups have questioned that ROS are produced upon TCR stimulation.ResultsTo shed some light onto this issue, we specifically measured superoxide production upon TCR ligation in primary human and mouse T lymphocytes. We showed that superoxide is indeed produced and released into the extracellular space. Antioxidants, such as superoxide dismutase and ascorbate, abolished superoxide production, but surprisingly did not affect activation, proliferation and cytokine secretion in TCR-stimulated primary human T cells. It has been suggested that T cells produce ROS via the NADPH oxidase 2 (NOX2). Therefore, we investigated whether T-cell activation is affected in NOX2-deficient mice (gp91 phox −/−). We found that T cells from these mice completely lack inducible superoxide production but display normal upregulation of activation markers and proliferation.ConclusionsCollectively, our data indicate that primary T cells produce extracellular superoxide upon TCR triggering, potentially via NOX2 at the plasma membrane. However, superoxide is not required for T-cell activation, proliferation and cytokine production.