Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential

2007 ◽  
Vol 292 (1) ◽  
pp. C148-C156 ◽  
Author(s):  
André Heinen ◽  
Amadou K. S. Camara ◽  
Mohammed Aldakkak ◽  
Samhita S. Rhodes ◽  
Matthias L. Riess ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Respiration ◽  
Channel Blocker ◽  
Electron Flow ◽  
Ros Generation ◽  
Ros Production ◽  
Guinea Pig Heart ◽  
Isolated Hearts ◽  
Channel Opening ◽  
Charged Membrane

We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca2+-sensitive K+ (mtKCa) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtKCa channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial K+ influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential (ΔΨm). To test this hypothesis, we measured effects of NS-1619, a putative mtKCa channel opener, and valinomycin, a K+ ionophore, on mitochondrial respiration, ΔΨm, and ROS generation in guinea pig heart mitochondria. NS-1619 (30 μM) increased state 2 and 4 respiration by 5.2 ± 0.9 and 7.3 ± 0.9 nmol O2·min−1·mg protein−1, respectively, with the NADH-linked substrate pyruvate and by 7.5 ± 1.4 and 11.6 ± 2.9 nmol O2·min−1·mg protein−1, respectively, with the FADH2-linked substrate succinate (+ rotenone); these effects were abolished by the mtKCa channel blocker paxilline. ΔΨm was not decreased by 10–30 μM NS-1619 with either substrate, but H2O2 release was increased by 44.8% (65.9 ± 2.7% by 30 μM NS-1619 vs. 21.1 ± 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H2O2 release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained ΔΨm, despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K+ influx during states 2 and 4 increases mitochondrial respiration while maintaining ΔΨm; this allows singlet electron uptake by O2 and ROS generation.

Mitochondrial (‘mild’) uncoupling and ROS production: physiologically relevant or not?

2011 ◽  
Vol 39 (5) ◽  
pp. 1305-1309 ◽  
Author(s):  
Irina G. Shabalina ◽  
Jan Nedergaard
Keyword(s):  
Membrane Potential ◽  
Oxidative Damage ◽  
Uncoupling Protein ◽  
Electron Flow ◽  
Ros Production ◽  
Present Evidence ◽  
Beneficial Effects ◽  
Reverse Electron Flow ◽  
Species Production ◽  
Mild Uncoupling

During the last decade, the possibility that ‘mild’ uncoupling could be protective against oxidative damage by diminishing ROS (reactive oxygen species) production has attracted much interest. In the present paper, we briefly examine the evidence for this possibility. It is only ROS production from succinate under reverse electron-flow conditions that is sensitive to membrane potential fluctuations, and so only this type of ROS production could be affected; however, the conditions under which succinate-supported ROS production is observed include succinate concentrations that are supraphysiological. Any decrease in membrane potential, even ‘mild uncoupling’, must necessarily lead to large increases in respiration, i.e. it must be markedly thermogenic. Mitochondria within cells are normally ATP-producing and thus already have a diminished membrane potential, and treatment of cells, organs or animals with small amounts of artificial uncoupler does not seem to have beneficial effects that are explainable via reduced ROS production. Although it has been suggested that members of the uncoupling protein family (UCP1, UCP2 and UCP3) may mediate a mild uncoupling, present evidence does not unequivocally support such an effect, e.g. the absence of the truly uncoupling protein UCP1 is not associated with increased oxidative damage. Thus present evidence does not support mild uncoupling as a physiologically relevant alleviator of oxidative damage.

Exercise training decreases rat heart mitochondria free radical generation but does not prevent Ca2+-induced dysfunction

2007 ◽  
Vol 102 (5) ◽  
pp. 1793-1798 ◽  
Author(s):  
Joseph W. Starnes ◽  
Brian D. Barnes ◽  
Marissa E. Olsen
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Electron Flow ◽  
Respiratory Control ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Calcium Overload ◽  
Ros Generation ◽  
Ros Production ◽  
Ptp Opening

Exercise provides cardioprotection against ischemia-reperfusion injury, a process involving mitochondrial reactive oxygen species (ROS) generation and calcium overload. This study tested the hypotheses that isolated mitochondria from hearts of endurance-trained rats have decreased ROS production and improved tolerance against Ca2+-induced dysfunction. Male Fischer 344 rats were either sedentary (Sed, n = 8) or endurance exercise trained (ET, n = 11) by running on a treadmill for 16 wk (5 days/wk, 60 min/day, 25 m/min, 6° grade). Mitochondrial oxidative phosphorylation measures were determined with glutamate-malate or succinate as substrates, and H2O2 production and permeability transition pore (PTP) opening were determined with succinate. All assays were carried out in the absence and presence of calcium. In response to 25 and 50 μM CaCl2, Sed and ET displayed similar decreases in state 3 respiration, respiratory control ratio, and ADP:O ratio. Ca2+-induced PTP opening was also similar. However, H2O2 production by ET was lower than Sed ( P < 0.05) in the absence of calcium (323 ± 12 vs. 362 ± 11 pmol·min−1·mg protein−1) and the presence of 50 μM CaCl2 (154 ± 3 vs. 197 ± 7 pmol·min−1·mg protein−1). Rotenone, which blocks electron flow from succinate to complex 1, reduced H2O2 production and eliminated differences between ET and Sed. Mitochondrial superoxide dismutase and glutathione peroxidase were not affected by exercise. Catalase activity was extremely low but increased 49% in ET ( P < 0.05). In conclusion, exercise reduces ROS production in myocardial mitochondria through adaptations specific to complex 1 but does not improve mitochondrial tolerance to calcium overload.

Mitochondrial matrix reactive oxygen species production is very sensitive to mild uncoupling

2003 ◽  
Vol 31 (6) ◽  
pp. 1300-1301 ◽  
Author(s):  
S. Miwa ◽  
M.D. Brand
Keyword(s):  
Reactive Oxygen Species ◽  
Membrane Potential ◽  
Complex I ◽  
Electron Flow ◽  
Ros Production ◽  
Oxygen Species ◽  
Inner Membrane ◽  
Reverse Electron Flow ◽  
The Matrix ◽  
Mild Uncoupling

Mitochondria produce ROS (reactive oxygen species) as a by-product of aerobic respiration. Several studies in mammals and birds suggest that the most physiologically relevant ROS production is from complex I following reverse electron flow, and is highly sensitive to membrane potential. A study of Drosophila mitochondria respiring glycerol 3-phosphate revealed that membrane potential-sensitive ROS production from complex I following reverse electron flow was on the matrix side of the inner membrane. A 10 mV decrease in membrane potential was enough to abolish around 70% of the ROS produced by complex I under these conditions. Another important ROS generator in this model, glycerol-3-phosphate dehydrogenase, produced ROS mostly to the cytosolic side; this ROS production was totally insensitive to a small decrease in membrane potential (10 mV). Thus mild uncoupling may be particularly significant for ROS production from complex I on the matrix side of the mitochondrial inner membrane.

scholarly journals Redox regulation of mitochondrial functional activity by quinones

2016 ◽  
Vol 103 (4) ◽  
pp. 439-458 ◽  
Author(s):  
NG Krylova ◽  
TA Kulahava ◽  
VT Cheschevik ◽  
IK Dremza ◽  
GN Semenkova ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Electron Transport ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Function ◽  
Mitochondrial Membrane ◽  
Reduction Potential ◽  
Electron Flow ◽  
Rat Liver Mitochondria ◽  
Ros Generation ◽  
Intact Cells

Quinones are among the rare compounds successfully used as therapeutic agents to correct mitochondrial diseases and as specific regulators of mitochondrial function within cells. The aim of the present study was to elucidate the redox-dependent effects of quinones on mitochondrial function. The functional parameters [respiratory activity, membrane potential, and reactive oxygen species (ROS) generation] of isolated rat liver mitochondria and mitochondria in intact cells were measured in the presence of eight exogenously applied quinones that differ in lipophilicity and one-electron reduction potential. The quinones affected the respiratory parameters of mitochondria, and dissipated the mitochondrial membrane potential as well as influenced (either decreased or enhanced) ROS generation, and restored the electron flow during electron transport chain inhibition. The stimulation of ROS production by juglone and 2,5-di-tert-butyl-1,4-benzoquinone was accompanied by a decrease in the acceptor control and respiration control ratios, dissipation of the mitochondrial membrane potential and induction of the reverse electron flow under succinate oxidation in isolated mitochondria. Menadione and 2,3,5-trimethyl-1,4-benzoquinone, which decreased the mitochondrial ROS generation, did not affect the mitochondrial potential and, vice versa, were capable of restoring electron transport during Complex I inhibition. In intact C6 cells, all the quinones, except for coenzyme Q10, decreased the mitochondrial membrane potential. Juglone, 1,4-benzoquinone, and menadione showed the most pronounced effects. These findings indicate that quinones with the reduction potential values E1/2 in the range from −99 to −260 mV were effective redox regulators of mitochondrial electron transport.

scholarly journals A1 adenosine receptor negatively modulates coronary reactive hyperemia via counteracting A2A-mediated H2O2 production and KATP opening in isolated mouse hearts

2013 ◽  
Vol 305 (11) ◽  
pp. H1668-H1679 ◽  
Author(s):  
Xueping Zhou ◽  
Bunyen Teng ◽  
Stephen Tilley ◽  
S. Jamal Mustafa
Keyword(s):  
Channel Blocker ◽  
Reactive Hyperemia ◽  
H2o2 Production ◽  
Wild Type ◽  
A1 Adenosine Receptor ◽  
Coronary Smooth Muscle ◽  
Isolated Hearts ◽  
System A ◽  
Channel Opening ◽  
Sch 58261

We previously demonstrated that A2A, but not A2B, adenosine receptors (ARs) mediate coronary reactive hyperemia (RH), possibly by producing H2O2 and, subsequently, opening ATP-dependent K+ (KATP) channels in coronary smooth muscle cells. In this study, A1 AR knockout (KO), A3 AR KO, and A1 and A3 AR double-KO (A1/A3 DKO) mice were used to investigate the roles and mechanisms of A1 and A3 ARs in modulation of coronary RH. Coronary flow of isolated hearts was measured using the Langendorff system. A1 KO and A1/A3 DKO, but not A3 KO, mice showed a higher flow debt repayment [∼30% more than wild-type (WT) mice, P < 0.05] following a 15-s occlusion. SCH-58261 (a selective A2A AR antagonist, 1 μM) eliminated the augmented RH, suggesting the involvement of enhanced A2A AR-mediated signaling in A1 KO mice. In isolated coronary arteries, immunohistochemistry showed an upregulation of A2A AR (1.6 ± 0.2 times that of WT mice, P < 0.05) and a higher magnitude of adenosine-induced H2O2 production in A1 KO mice (1.8 ± 0.3 times that of WT mice, P < 0.05), which was blocked by SCH-58261. Catalase (2,500 U/ml) and glibenclamide (a KATP channel blocker, 5 μM), but not NG-nitro-l-arginine methyl ester, also abolished the enhanced RH in A1 KO mice. Our data suggest that A1, but not A3, AR counteracts the A2A AR-mediated CF increase and that deletion of A1 AR results in upregulation of A2A AR and/or removal of the negative modulatory effect of A1 AR, thus leading to an enhanced A2A AR-mediated H2O2 production, KATP channel opening, and coronary vasodilation during RH. This is the first report implying that A1 AR has a role in coronary RH.

scholarly journals Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation–reduction state

2002 ◽  
Vol 368 (2) ◽  
pp. 545-553 ◽  
Author(s):  
Yulia KUSHNAREVA ◽  
Anne N. MURPHY ◽  
Alexander ANDREYEV
Keyword(s):  
Reactive Oxygen Species ◽  
Cytochrome C ◽  
Complex I ◽  
Electron Flow ◽  
Reactive Oxygen ◽  
Complex Iii ◽  
Ros Generation ◽  
Ros Production ◽  
Oxygen Species ◽  
Reduced State

Several lines of evidence indicate that mitochondrial reactive oxygen species (ROS) generation is the major source of oxidative stress in the cell. It has been shown that ROS production accompanies cytochrome c release in different apoptotic paradigms, but the site(s) of ROS production remain obscure. In the current study, we demonstrate that loss of cytochrome c by mitochondria oxidizing NAD+-linked substrates results in a dramatic increase of ROS production and respiratory inhibition. This increased ROS production can be mimicked by rotenone, a complex I inhibitor, as well as other chemical inhibitors of electron flow that act further downstream in the electron transport chain. The effects of cytochrome c depletion from mitoplasts on ROS production and respiration are reversible upon addition of exogenous cytochrome c. Thus in these models of mitochondrial injury, a primary site of ROS generation in both brain and heart mitochondria is proximal to the rotenone inhibitory site, rather than in complex III. ROS production at complex I is critically dependent upon a highly reduced state of the mitochondrial NAD(P)+ pool and is achieved upon nearly complete inhibition of the respiratory chain. Redox clamp experiments using the acetoacetate/d-β-hydroxybutyrate couple in the presence of a maximally inhibitory rotenone concentration suggest that the site is approx. 50mV more electronegative than the NADH/NAD+ couple. In the absence of inhibitors, this highly reduced state of mitochondria can be induced by reverse electron flow from succinate to NAD+, accounting for profound ROS production in the presence of succinate. These results lead us to propose a model of thermodynamic control of mitochondrial ROS production which suggests that the ROS-generating site of complex I is the Fe—S centre N-1a.

Essential role of complex II of the respiratory chain in hypoxia-induced ROS generation in the pulmonary vasculature

2003 ◽  
Vol 284 (5) ◽  
pp. L710-L719 ◽  
Author(s):  
Renate Paddenberg ◽  
Barat Ishaq ◽  
Anna Goldenberg ◽  
Petra Faulhammer ◽  
Frank Rose ◽  
...  
Keyword(s):  
Succinate Dehydrogenase ◽  
Essential Role ◽  
Electron Flow ◽  
Enzymatic Reaction ◽  
Fumarate Reductase ◽  
Pulmonary Vasculature ◽  
Ros Generation ◽  
Ros Production ◽  
Complex Ii ◽  
Vascular Walls

In the pulmonary vasculature, the mechanisms responsible for oxygen sensing and the initiation of hypoxia-induced vasoconstriction and vascular remodeling are still unclear. Nitric oxide (NO) and reactive oxygen species (ROS) are discussed as early mediators of the hypoxic response. Here, we describe a quantitative analysis of NO- and ROS-producing cells within the vascular walls of murine lung sections cultured at normoxia or hypoxia. Whereas the number of NO-producing cells was not changed by hypoxia, the number of ROS-generating cells was significantly increased. Addition of specific inhibitors revealed that mitochondria were the source of ROS. The participation of the individual mitochondrial complexes differed in normoxic and hypoxic ROS generation. Whereas normoxic ROS production required complexes I and III, hypoxic ROS generation additionally demanded complex II. Histochemically demonstrable succinate dehydrogenase activity of complex II in the arterial wall decreased during hypoxia. Inhibition of the reversed enzymatic reaction, i.e., fumarate reductase, by application of succinate, specifically abolished hypoxic, but not normoxic, ROS generation. Thus complex II plays an essential role in hypoxic ROS production. Presumably, its catalytic activity switches from succinate dehydrogenase to fumarate reductase at reduced oxygen tension, thereby modulating the directionality of the electron flow.

scholarly journals Auraptene Mitigates Parkinson’s Disease-Like Behavior by Protecting Inhibition of Mitochondrial Respiration and Scavenging Reactive Oxygen Species

2019 ◽  
Vol 20 (14) ◽  
pp. 3409 ◽  
Author(s):  
Yunseon Jang ◽  
Hyosun Choo ◽  
Min Joung Lee ◽  
Jeongsu Han ◽  
Soo Jeong Kim ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Substantia Nigra ◽  
Mitochondrial Respiration ◽  
Dopaminergic Neurons ◽  
Reactive Oxygen ◽  
Ros Generation ◽  
Ros Production ◽  
Oxygen Species

Current therapeutics for Parkinson’s disease (PD) are only effective in providing relief of symptoms such as rigidity, tremors and bradykinesia, and do not exert disease-modifying effects by directly modulating mitochondrial function. Here, we investigated auraptene (AUR) as a potent therapeutic reagent that specifically protects neurotoxin-induced reduction of mitochondrial respiration and inhibits reactive oxygen species (ROS) generation. Further, we explored the mechanism and potency of AUR in protecting dopaminergic neurons. Treatment with AUR significantly increased the viability of substantia nigra (SN)-derived SN4741 embryonic dopaminergic neuronal cells and reduced rotenone-induced mitochondrial ROS production. By inducing antioxidant enzymes AUR treatment also increased oxygen consumption rate. These results indicate that AUR exerts a protective effect against rotenone-induced mitochondrial oxidative damage. We further assessed AUR effects in vivo, investigating tyrosine hydroxylase (TH) expression in the striatum and substantia nigra of MPTP-induced PD model mice and behavioral changes after injection of AUR. AUR treatment improved movement, consistent with the observed increase in the number of dopaminergic neurons in the substantia nigra. These results demonstrate that AUR targets dual pathogenic mechanisms, enhancing mitochondrial respiration and attenuating ROS production, suggesting that the preventative potential of this natural compound could lead to improvement in PD-related neurobiological changes.

Reverse electron flow-induced ROS production is attenuated by activation of mitochondrial Ca2+-sensitive K+ channels

2007 ◽  
Vol 293 (3) ◽  
pp. H1400-H1407 ◽  
Author(s):  
André Heinen ◽  
Mohammed Aldakkak ◽  
David F. Stowe ◽  
Samhita S. Rhodes ◽  
Matthias L. Riess ◽  
...  
Keyword(s):  
Complex I ◽  
Redox State ◽  
Channel Blocker ◽  
Electron Flow ◽  
Release Rates ◽  
Guinea Pig Heart ◽  
Low Concentrations ◽  
Transport Chain ◽  
Reverse Electron Flow ◽  
Flow Matrix

Mitochondria generate reactive oxygen species (ROS) dependent on substrate conditions, O2 concentration, redox state, and activity of the mitochondrial complexes. It is well known that the FADH2-linked substrate succinate induces reverse electron flow to complex I of the electron transport chain and that this process generates superoxide (O2•−); these effects are blocked by the complex I blocker rotenone. We demonstrated recently that succinate + rotenone-dependent H2O2 production in isolated mitochondria increased mildly on activation of the putative big mitochondrial Ca2+-sensitive K+ channel (mtBKCa) by low concentrations of 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2 H-benzimidazol-2-one (NS-1619). In the present study we examined effects of NS-1619 on mitochondrial O2 consumption, membrane potential (ΔΨm), H2O2 release rates, and redox state in isolated guinea pig heart mitochondria respiring on succinate but without rotenone. NS-1619 (30 μM) increased state 2 and state 4 respiration by 26 ± 4% and 14 ± 4%, respectively; this increase was abolished by the BKCa channel blocker paxilline (5 μM). Paxilline alone had no effect on respiration. NS-1619 did not alter ΔΨm or redox state but decreased H2O2 production by 73% vs. control; this effect was incompletely inhibited by paxilline. We conclude that under substrate conditions that allow reverse electron flow, matrix K+ influx through mtBKCa channels reduces mitochondrial H2O2 production by accelerating forward electron flow. Our prior study showed that NS-1619 induced an increase in H2O2 production with blocked reverse electron flow. The present results suggest that NS-1619-induced matrix K+ influx increases forward electron flow despite the high reverse electron flow, and emphasize the importance of substrate conditions on interpretation of effects on mitochondrial bioenergetics.

scholarly journals Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors Induce Oxidative Stress in Patient-Derived Glioblastoma Cells

2021 ◽  
Vol 11 (7) ◽  
pp. 884
Author(s):  
Ana Kostić ◽  
Sofija Jovanović Stojanov ◽  
Ana Podolski-Renić ◽  
Marija Nešović ◽  
Miodrag Dragoj ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Kinase Inhibitors ◽  
Ros Generation ◽  
Ros Production ◽  
Dna Breaks ◽  
Src Tyrosine Kinase

Background: Glioblastoma (GBM) highly expresses Src tyrosine kinase involved in survival, proliferation, angiogenesis and invasiveness of tumor cells. Src activation also reduces reactive oxygen species (ROS) generation, whereas Src inhibitors are able to increase cellular ROS levels. Methods: Pro-oxidative effects of two pyrazolo[3,4-d]pyrimidine derivatives—Src tyrosine kinase inhibitors, Si306 and its prodrug pro-Si306—were investigated in human GBM cells U87 and patient-derived GBM-6. ROS production and changes in mitochondrial membrane potential were assessed by flow cytometry. The expression levels of superoxide dismutase 1 (SOD1) and 2 (SOD2) were studied by Western blot. DNA damage, cell death induction and senescence were also examined in GBM-6 cells. Results: Si306 and pro-Si306 more prominently triggered ROS production and expression of antioxidant enzymes in primary GBM cells. These effects were followed by mitochondrial membrane potential disruption, double-strand DNA breaks and senescence that eventually led to necrosis. Conclusion: Src kinase inhibitors, Si306 and pro-Si306, showed significant pro-oxidative potential in patient-derived GBM cells. This feature contributes to the already demonstrated anti-glioblastoma properties of these compounds in vitro and in vivo and encourages clinical investigations.

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