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NADPH oxidases are actively consuming O2 and producing H2O2 in freshly isolated synaptosomes. Successive injections of 200 μM deoxygenated NADPH batches on synaptosomes triggered reproducible oxygen consumption (a, b) and H2O2 production as detected simultaneously by HRP/Amplex Red fluorescence (c, d) or using electrochemical sensor (e, f). The effects of inclusion of the NOX inhibitors Vas-2870 (10 μM) (a, c, e) or ebselen (10 μM) (b, d, f) are shown. (g) Quantifications of the overall O2 and H2O2 fluxes induced by the three added doses of NADPH. The two methods of H2O2 detection yielded similar results. Both of the added NOX inhibitors, VAS-2870 (10 μM) and Ebselen (10 μM), caused significant inhibition of NADPH-triggered oxygen flux (h) and H2O2 flux (i). Values are given as mean ± SEM, paired Student t -test was used for paired groups to determine statistical significance in comparison with NADPH-induced activity, p ∗ < 0.05 , and n = 5 – 8 .
Source publication
Disruption of cellular redox homeostasis is implicated in a wide variety of pathologic conditions and aging. A fundamental factor that dictates such balance is the ratio between mitochondria-mediated complete oxygen reduction into water and incomplete reduction into superoxide radical by mitochondria and NADPH oxidase (NOX) enzymatic activity. Here...
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NADPH oxidase (NOX) enzymes are involved in a various physiological and pathological processes such as platelet activation and inflammation. Interestingly, we found that the pan-NOX inhibitors VAS compounds (VAS2870 and its analog VAS3947) exerted a highly potent antiplatelet effect. Unlike VAS compounds, concurrent inhibition of NOX1, 2, and 4 by...
Citations
... Tissuedependent homogenization was then carried out according to the manufacturer's instructions. The Oxygraph-2k (O2k, OROBOROS Instruments, Innsbruck, Austria) was used for measurements of flux of oxygen, electron transport (ET) capacity and OXPHOS capacity in 2-mL chambers (Abdel-Rahman et al., 2016). Before starting the experiment, calibration at air saturation versus zero oxygen was performed by allowing the respiration medium (MIR05 buffer), the buffer to equilibrate with air in the oxygraph chambers and stirred at 540-560 rpm for 30-40 min, until a stable signal was detected. ...
Sevoflurane (Sev) is one of the commonly used inhalation anesthetic chemicals in clinics. It has great impact on spermatogenesis and fertilization in male animals. The underlying mechanism remains largely unexplored. Based on our previous research, we hypothesized that Sev induced iron metabolism disturbance in the testis and epididymis and inhibited the spermatogenesis. In this study, two-month-old C57BL/6 male mice were treated with 3% Sev for 6 h, and their fertility (including sperm concentration, sperm mobility, and the number of offspring) was evaluated. Mice testis, epididymis, and sperm were harvested and subjected to Western blot analysis and immunofluorescence analysis. Iron levels were reflected by the gene expression of iron metabolism-related proteins (including ferritin, TfR1, and FpN1) and ICP-MS and Perl’s iron staining. Electron transport and oxidative phosphorylation levels were measured by Oxygraph-2k and ATP contents. The activity of ribonucleotide reductase was evaluated by assay kit. DNA synthesis status in testis and/or epididymis was marked with BrdU. Cell proliferation was evaluated by double immunofluorescence staining of specific protein marker expression. Our results revealed that the mice exposed to Sev showed damaged testicular and epididymis structure and significantly reduced the sperm concentration, sperm motility, and fertility. Sev decreases the iron levels through down-regulating the expression of H-ferritin, L-ferritin, and FpN1, and up-regulating the expression of TfR1 in the testis and epididymis. Iron levels also significantly reduced in germ cells which decrease the number of germ cells, including sperm, Sertoli cells, and primary spermatocyte. Iron deficiency not only decreases electron transport, oxidative phosphorylation level, and ATP production but also suppresses the activity of ribonucleotide reductase and the expression of Ki67, DDX4, GATA1, and SCP3, indicating that Sev affects the spermatogenesis and development. Meanwhile, Sev impaired the blood-testis barrier by decreasing the ZO1 expression in the testis and epididymis. The damage effect induced by Sev can be significantly ameliorated by iron supplementation. In conclusion, our study illustrates a new mechanism by which Sev inhibits spermatogenesis and fertility through an oxidative phosphorylation pathway due to iron deficiency of epididymis and testis or sperm. Furthermore, the damaging effects could be ameliorated by iron supplementation.
... As previously described, the high-resolution Oroboros Oxygraph 2K (Oroboros Instruments, Innsbruck, Austria) was used to assess mitochondrial respiration rates in bEnd.3 cells using two 2-ml chambers under continuous stirring at 37°C (Abdel-Rahman et al., 2016;Roy Chowdhury et al., 2020). The treated bEnd.3 cells were washed with PBS and then suspended in DMEM medium and transferred into the O2k chambers. ...
Several studies have demonstrated the protective effect of dl-3-n-Butylphthalide (NBP) against cerebral ischemia, which may be related to the attenuation of mitochondrial dysfunction. However, the specific mechanism and targets of NBP in cerebral ischemia/reperfusion remains unclear. In this study, we used a chemical proteomics approach to search for targets of NBP and identified cytochrome C oxidase 7c (Cox7c) as a key interacting target of NBP. Our findings indicated that NBP inhibits mitochondrial apoptosis and reactive oxygen species (ROS) release and increases ATP production through upregulation of Cox7c. Subsequently, mitochondrial respiratory capacity was improved and the HIF-1α/VEGF pathway was upregulated, which contributed to the maintenance of mitochondrial membrane potential and blood brain barrier integrity and promoting angiogenesis. Therefore, our findings provided a novel insight into the mechanisms underlying the neuroprotective effects of NBP, and also proposed for the first time that Cox7c exerts a critical role by protecting mitochondrial function.
... Piccoli et al. (2005) reported that NADPH oxidase isoforms 2 and 4 consumed up to 40% of total oxygen consumption in CD34+ human hemopoietic stem cells (Piccoli et al., 2005), yet these cells are cultured at non-physiological oxygen levels limiting the translation of their findings in vivo. On the contrary, another study reported that utilization of oxygen from NADPH oxidases accounted for less than 1% of total oxygen consumption in synaptosomes (Abdel-Rahman et al., 2016). Based on these fragmented data, it becomes clear that the widely held view that mitochondrial respiration consumes over 95% of the oxygen should be re-evaluated and always put in context (e.g., cell type, condition) (Hill et al., 2012;Pittman, 2016). ...
Oxygen, a stable di-radical, is critical for aerobic life, and a mismatch between its supply and demand during exercise decreases performance and precipitates fatigue. In this chapter, we highlight the critical redox components (i.e., signaling as well as structural and functional modifications mediated by reactive oxygen and nitrogen species) at each step of the oxygen pathway from atmospheric air to muscle mitochondria. We split the oxygen pathway into five major “stages”: (1) lungs, (2) erythrocytes, (3) microcirculation, (4) muscle and (5) mitochondria. We present evidence that redox mechanisms regulate oxygen transport throughout. Thus, oxygen transport can no longer be considered a redox neutral area. Following a quantitative approach, we emphasize the need to implement more spatiotemporally refined redox interventions to increase oxygen delivery and improve exercise performance. The architectural and numerical basis of oxygen transport can inform experimentally testable predictions in redox biology, exercise physiology and sports nutrition.
... EtOH exposure can enhance ROS production via induction of NADPH oxidases (NOXs) [23,24], its CYP2E1-catalyzed metabolism [25], and via the regeneration of NAD + for alcohol and aldehyde dehydrogenase activity [26][27][28]. NOXs produce superoxide anions [29], which participate in neural cellular signaling and in the immune response to pathogens [30,31]. EtOH-induced ROS enhancement can contribute to multiple cellular effects implicated in FASD mechanisms, including DNA damage [26], epigenetic changes [32][33][34], altered protein levels and modifications affecting signal transduction pathways [35,36] and cellular processes (e. g. apoptosis) [32]. ...
The breast cancer 1 protein (BRCA1) facilitates DNA repair, preventing embryolethality and protecting the fetus from reactive oxygen species (ROS)-induced developmental disorders mediated by oxidatively damaged DNA. Alcohol (ethanol, EtOH) exposure during pregnancy causes fetal alcohol spectrum disorders (FASD), characterized by aberrant behaviour and enhanced ROS formation and proteasomal protein degradation. Herein, ROS-producing NADPH oxidase (NOX) activity was higher in Brca1 ± vs. +/+ fetal and adult brains, and further enhanced by a single EtOH exposure. EtOH also enhanced catalase and proteasomal activities, while conversely reducing BRCA1 protein levels without affecting Brca1 gene expression. EtOH-initiated adaptive postnatal freezing behaviour was lost in Brca1 ± progeny. Pretreatment with the free radical spin trap and ROS inhibitor phenylbutylnitrone blocked all EtOH effects, suggesting ROS-dependent mechanisms. This is the first in vivo evidence of NOX regulation by BRCA1, and of EtOH-induced, ROS-mediated depletion of BRCA1, revealing novel mechanisms of BRCA1 protection in FASD.
... Mitochondrial respiratory assessment and hydrogen peroxide production were carried out at 37°C using the high-resolution respirometry system O2k (Oroboros Instruments, Innsbruck, Austria) in 2-ml chambers as previously described [24,25]. Before starting the experiment, calibration at air saturation versus zero oxygen was performed by allowing the respiration medium, MIR05 to equilibrate with air in the oxygraph chambers and stirred at 540-560 rpm for 30-40 min, until a stable signal was detected. ...
... Mitochondrial respiration and ROS generation are interconnected [24,25,64]. In the fed state, a decrease in mitochondrial OXPHOS may result in highly reduced ETC components, which are more likely to leak electrons to molecular oxygen [26,65]. ...
Introduction
Incidents of myocardial infarction and sudden cardiac arrest vary with time of the day, but the mechanism for this effect is not clear. We hypothesized that diurnal changes in the ability of cardiac mitochondria to control calcium homeostasis dictate vulnerability to cardiovascular events.
Objectives
Here we investigate mitochondrial calcium dynamics, respiratory function, and reactive oxygen species (ROS) production in mouse heart during different phases of wake versus sleep periods.
Methods
We assessed time-of-the-day dependence of calcium retention capacity of isolated heart mitochondria from young male C57BL6 mice. Rhythmicity of mitochondrial-dependent oxygen consumption, ROS production and transmembrane potential in homogenates were explored using the Oroboros O2k Station equipped with a fluorescence detection module. Changes in expression of essential clock and calcium dynamics genes/proteins were also determined at sleep versus wake time points.
Results
Our results demonstrate that cardiac mitochondria exhibit higher calcium retention capacity and higher rates of calcium uptake during sleep period. This was associated with higher expression of clock gene Bmal1, lower expression of per2, greater expression of MICU1 gene (mitochondrial calcium uptake 1), and lower expression of the mitochondrial transition pore regulator gene cyclophilin D. Protein levels of mitochondrial calcium uniporter (MCU), MICU2, and sodium/calcium exchanger (NCLX) were also higher at sleep onset relative to wake period. While complex I and II-dependent oxygen utilization and transmembrane potential of cardiac mitochondria were lower during sleep, ROS production was increased presumably due to mitochondrial calcium sequestration.
Conclusions
Taken together, our results indicate that retaining mitochondrial calcium in the heart during sleep dissipates membrane potential, slows respiratory activities, and increases ROS levels, which may contribute to increased vulnerability to cardiac stress during sleep-wake transition. This pronounced daily oscillations in mitochondrial functions pertaining to stress vulnerability may at least in part explain diurnal prevalence of cardiac pathologies.
... We also examined the activity of phagocytic NOX2 in circulating neutrophils freshly isolated from all groups. To accomplish these goals, we used high resolution respirometry to monitor oxygen consumption in isolated platelets, and combined high resolution respirometry with fluorometry to simultaneously monitor NOX-dependent oxygen consumption and hydrogen peroxide (H 2 O 2 ) production [21] in isolated neutrophils. Spin-trapping electron paramagnetic resonance (EPR) spectroscopy has also been used for monitoring NOXdependent superoxide bursts by neutrophils. ...
... Determination of NOX activity in neutrophils by oroboros high resolution O 2 k oxygraph NOX activity in neutrophils' preparations was determined by measuring, in the same sample, NADPHinduced oxygen consumption and the associated rate of H 2 O 2 formation simultaneously using Amplex Red fluorescence as described [21]. Neutrophils were added to the respiration medium in the chamber. ...
Mitochondrial and immune dysfunctions are often implicated in the aetiology of autism spectrum disorder (ASD). Here, we studied for the first time the relationship between ASD severity measures and mitochondrial respiratory rates in freshly isolated platelets as well as the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in isolated neutrophils. We also verified the impact of hyperbaric oxygen therapy (HBOT) on mitochondrial and immune functions as well as on ASD severity measures. Blood samples were collected from three age-matched male groups (Control (Norm-N), autistic (Aut-N), and autistic + HBOT (Aut-H); N = 10 per group). Using high resolution respirometry, we found that routine basal respiration, complex I- and complex I + II-dependent oxidative phosphorylation rate were significantly impaired in Aut-N platelets. Similarly, deficits in immune response of neutrophils were evidenced through lower rates of oxygen consumption and reactive oxygen species (ROS) production by phagocytic NOX. ASD-related behavioural outcomes were found to moderately correlate with platelets’ mitochondrial bioenergetic parameters as well as with NOX-mediated activity in neutrophils. HBOT was not able to improve mitochondrial dysfunctions or to counteract ASD-related behavioral deficits. Although HBOT improved one measure of the immune response; namely, NOX-mediated superoxide burst, this was not associated with significant changes in trends of recurrent infections between groups. Taken together, our data suggest that ASD-associated mitochondria and immune deficits are detectable in platelets and neutrophils. We also found no evidence that HBOT confers any significant improvement of ASD-associated physiological or behavioural phenotypes.
... Therefore, TLR4 antagonist reduces the infarct size, attenuates IS-induced inflammatory changes, and I/R injury. [42,43] Different in vitro and in vivo studies illustrated that VPN inhibits I/R injury in IS through suppression of TLR4 receptors and NF-κB signaling pathway in animal model studies. [44] Neuronal mitochondrial reactive oxygen species contribute to the pathogenesis of I/R injury in IS as well as neurodegeneration and glutamate excitotoxicity. ...
Vinpocetine (VPN) is a synthetic ethyl-ester derivative of the alkaloid apovincamine from Vinca minor leaves. VPN is a selective inhibitor of phosphodiesterase type 1 (PDE1) that has potential neurological effects through inhibition of voltage-gated sodium channel and reduction of neuronal calcium influx. VPN has noteworthy antioxidant, anti-inflammatory, and anti-apoptotic effects with inhibitory effect on glial and astrocyte cells during and following ischemic stroke (IS). VPN is effective as adjuvant therapy in the management of epilepsy; it reduces seizure frequency by 50% in a dose of 2 mg/kg/day. VPN improves psychomotor performances through modulation of brain monoamine pathway mainly on dopamine and serotonin, which play an integral role in attenuation of depressive symptoms. VPN recover cognitive functions and spatial memory through inhibition of hippocampal and cortical PDE1 with augmentation of cyclic adenosin monophosphate and cyclic guanosin monophosphate ratio, enhancement of cholinergic neurotransmission, and inhibition of neuronal inflammatory mediators. Therefore, VPN is an effective agent in the management of IS and plays an integral role in the prevention and attenuation of poststroke epilepsy, depression, and cognitive deficit through direct cAMP/cGMP-dependent pathway or indirectly through anti-inflammatory and antioxidant effects.
... In addition, injured neurons in IS release specific proteins called danger associated molecular patterns including; heat shock protein (HSP), high mobility groupbox 1 protein (HMGB-1), ATP and nicotinamide adenine dinucleotide (NAD) which activate TLR4 receptors on perivascular macrophage, microglia and endothelial cells. Therefore, TLR4 antagonist reduces infarct size attenuates IS-induced inflammatory changes and I/R injury [37,38]. Different in vitro and in vivo studies illustrated that VPN inhibits I/R injury in IS through suppression of TLR4 receptors and NF-K B signaling pathway in animal model studies [39]. ...
Vinpocetine (VPN) is a synthetic ethyl-ester derivative of the alkaloid apovincamine from Vinca minor leaves. VPN is a selective inhibitor of phospho-diesterase type 1 (PDE1) has potential neurological effects through inhibition of voltage gated sodium channel and reduction of neuronal calcium influx. VPN have noteworthy antioxidant, anti-inflammatory and anti-apoptotic effects with inhibitory effect on glial and astrocyte cells during and following ischemic stroke (IS). VPN is effective as an adjuvant therapy in the management of epilepsy; it reduces seizure frequency by 50% in a dose of 2 mg/kg/day. VPN improves psychomotor performances through modulation of brain monoamine pathway mainly on dopamine and serotonin, which play an integral role in attenuation of depressive symptoms. VPN recover cognitive functions and spatial memory through inhibition of hippocampal and cortical PDE-1with augmentation of cAMP/cGMP ratio, enhancement of cholinergic neurotransmission and inhibition of neuronal inflammatory mediators. Therefore, VPN is an effective agent in the management of ischemic stroke and plays an integral role in the prevention and attenuation of post-stroke epilepsy, depression and cognitive deficit through direct cAMP/cGMP-dependent pathway or indirectly through anti-inflammatory and anti-oxidant effects.
... In addition, injured neurons in IS release specific proteins called danger associated molecular patterns including; heat shock protein (HSP), high mobility groupbox 1 protein (HMGB-1), ATP and nicotinamide adenine dinucleotide (NAD) which activate TLR4 receptors on perivascular macrophage, microglia and endothelial cells. Therefore, TLR4 antagonist reduces infarct size attenuates IS-induced inflammatory changes and I/R injury [37,38]. Different in vitro and in vivo studies illustrated that VPN inhibits I/R injury in IS through suppression of TLR4 receptors and NF-K B signaling pathway in animal model studies [39]. ...
Vinpocetine (VPN) is a synthetic ethyl-ester derivative of the alkaloid apovincamine from Vinca minor leaves. VPN is a selective inhibitor of phosphodiesterase type 1 (PDE1) has potential neurological effects through inhibition of voltage gated sodium channel and reduction of neuronal calcium influx. VPN have noteworthy antioxidant, anti-inflammatory and anti-apoptotic effects with inhibitory effect on glial and astrocyte cells during and following ischemic stroke (IS). VPN is effective as an adjuvant therapy in the management of epilepsy; it reduces seizure frequency by 50% in a dose of 2 mg/kg/day. VPN improves psychomotor performances through modulation of brain monoamine pathway mainly on dopamine and serotonin, which play an integral role in attenuation of depressive symptoms. VPN recover cognitive functions and spatial memory through inhibition of hippocampal and cortical PDE-1with augmentation of cAMP/cGMP ratio, enhancement of cholinergic neurotransmission and inhibition of neuronal inflammatory mediators. Therefore, VPN is an effective agent in the management of ischemic stroke and plays an integral role in the prevention and attenuation of post-stroke epilepsy, depression and cognitive deficit through direct cAMP/cGMP-dependent pathway or indirectly through anti-inflammatory and anti-oxidant effects.
... Oxygen consumption of synaptosomes is attributed to the substrate oxidation of in situ mitochondria [47]. Thus, glucose used as an energy supply for synaptosomes is metabolised in glycolysis and subsequently in the mitochondria. ...
... The information are indirect, because (i) ROS sources other than mitochondria e.g. NADPH oxidases [47,48], and nitrogen monoxide synthase (NOS) [49] can also be found in synaptosomes (ii) the cytoplasm possesses many enzymatic and nonenzymatic ROS scavenging systems [50,51] and our detection system for H 2 O 2 is extra-synaptosomal; in synaptosomes between the mitochondrial membranes and the plasma membrane of the synaptosomes both ROS generating and scavenging mechanisms can be found, therefore more direct information was acquired using isolated mitochondria. ...
Vinpocetine is considered as neuroprotectant drug and used for treatment of brain ischemia and cognitive deficiencies for decades. A number of enzymes, channels and receptors can bind vinpocetine, however the mechanisms of many effects’ are still not clear. The present study investigated the effects of vinpocetine from the mitochondrial bioenergetic aspects. In primary brain capillary endothelial cells the purinergic receptor-stimulated mitochondrial Ca²⁺ uptake and efflux were studied. Vinpocetine exerted a partial inhibition on the mitochondrial calcium efflux. In rodent brain synaptosomes vinpocetine (30 μM) inhibited respiration in uncoupler stimulated synaptosomes and decreased H2O2 release from the nerve terminals in resting and in complex I inhibited conditions, respectively. In isolated rat brain mitochondria using either complex I or complex II substrates leak respiration was stimulated, but ADP-induced respiration was inhibited by vinpocetine. The stimulation of oxidation was associated with a small extent of membrane depolarization. Mitochondrial H2O2 production was inhibited by vinpocetine under all conditions investigated. The most pronounced effects were detected with the complex II substrate succinate. Vinpocetine also mitigated both Ca²⁺-induced mitochondrial Ca²⁺-release and Ca²⁺-induced mitochondrial swelling. It lowered the rate of mitochondrial ATP synthesis, while increasing ATPase activity. These results indicate more than a single mitochondrial target of this vinca alkaloid. The relevance of the affected mitochondrial mechanisms in the anti ischemic effect of vinpocetine is discussed.
