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PloS One 2016The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen...
The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore-induced maximal OCR may be associated with impaired metabolism of mitochondrial respiratory substrates.
Topics: Cell Line, Tumor; Cell Respiration; Electron Transport; Humans; Mitochondria; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption
PubMed: 26950698
DOI: 10.1371/journal.pone.0150967 -
Neuroreport Oct 2022Strokes represent as one of the leading causes of death and disability in the USA, however, there is no optimal treatment to reduce the occurrence or improve prognosis....
OBJECTIVE
Strokes represent as one of the leading causes of death and disability in the USA, however, there is no optimal treatment to reduce the occurrence or improve prognosis. Preconditioning of tissues triggers ischemic tolerance, a physiological state that may involve a metabolic switch (i.e. from glycolysis to oxidative phosphorylation or OxPhos) to preserve tissue viability under an ischemic insult. Here, we hypothesized that metabolic switching of energy source from glucose to galactose in cultured mesenchymal stem cells (MSCs) stands as an effective OxPhos-enhancing strategy.
METHODS
MSCs were grown under ambient condition (normal MSCs) or metabolic switching paradigm (switched MSCs) and then assayed for oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) using the Seahorse technology to assess mitochondrial respiration.
RESULTS
Normal MSCs showed a lower OCR/ECAR ratio than switched MSCs at baseline (P < 0.0001), signifying that there were greater levels of OxPhos compared to glycolysis in switched MSCs. By modulating the mitochondrial metabolism with oligomycin (time points 4-6), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (7-9), and rotenone and antimycin (time points 10-12), switched MSCs greater reliance on OxPhos was further elucidated (time points 5-12; P < 0.0001; time point 4; P < 0.001).
CONCLUSION
The metabolic switch from glycolytic to oxidative metabolism amplifies the OxPhos potential of MSCs, which may allow these cells to afford more robust therapeutic effects against neurological disorders that benefit from ischemic tolerance.
Topics: Galactose; Glucose; Glycolysis; Mesenchymal Stem Cells; Oligomycins; Oxidative Phosphorylation; Rotenone
PubMed: 36126260
DOI: 10.1097/WNR.0000000000001828 -
FEBS Letters Feb 1994In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycin A and the highly specific mitochondrial ATP-synthase inhibitor oligomycin...
In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycin A and the highly specific mitochondrial ATP-synthase inhibitor oligomycin are shown to induce an apoptotic suicide response in cultured human lymphoblastoid and other mammalian cells within 12-18 h. The mitochondrial inhibitors do not induce apoptosis in cells depleted of mitochondrial DNA and thus lacking an intact mitochondrial respiratory chain. Apoptosis induced by respiratory chain inhibitors is not inhibited by the presence of Bcl-2. We discuss the possible role of mitochondrial induced apoptosis in the ageing process and age-associated diseases.
Topics: Animals; Antimycin A; Apoptosis; Cell Nucleus; Culture Media; DNA; Energy Metabolism; Humans; Leukemia; Melanoma; Mice; Oligomycins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rotenone; Tumor Cells, Cultured
PubMed: 8313978
DOI: 10.1016/0014-5793(94)80380-3 -
The Biochemical Journal Jul 19681. The effects of dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enzyme systems related to respiratory-chain phosphorylation were compared.... (Comparative Study)
Comparative Study
1. The effects of dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enzyme systems related to respiratory-chain phosphorylation were compared. Dicyclohexylcarbodi-imide and oligomycin A have very similar functional effects, giving 50% inhibition of ATP-utilizing and ATP-generating systems at concentrations below 0.8nmole/mg. of submitochondrial-particle protein. Aurovertin is a more potent inhibitor of ATP synthesis, giving 50% inhibition at 0.2nmole/mg. of protein. However, aurovertin is a less potent inhibitor of ATP-utilizing systems: the ATP-driven energy-linked nicotinamide nucleotide transhydrogenase is 50% inhibited at 3.0nmoles/mg. of protein and the ATP-driven reduction of NAD(+) by succinate is 50% inhibited at 0.95nmole/mg. of protein. 2. With EDTA-particles (prepared by subjecting mitochondria to ultrasonic radiation at pH9 in the presence of 2mm-EDTA) the maximum stimulation of the ATP-driven partial reactions is effected by similar concentrations of oligomycin A and dicylcohexylcarbodi-imide, but the latter is less effective. The stimulatory effects of suboptimum concentrations of dicyclohexylcarbodi-imide and oligomycin A are additive. Aurovertin does not stimulate these reactions or interfere with the stimulation by the other inhibitors. 3. Dicyclohexylcarbodi-imide and oligomycin A stimulate the aerobic energy-linked nicotinamide nucleotide transhydrogenase of EDTA-particles, but the optimum concentration is higher than that required for the ATP-driven partial reactions. Aurovertin has no effect on this reaction. 4. The site of action of dicyclohexylcarbodi-imide is in CF(0), the mitochondrial fraction that confers oligomycin sensitivity on F(1) mitochondrial adenosine triphosphatase.
Topics: Adenine Nucleotides; Adenosine Triphosphatases; Animals; Antimetabolites; Cattle; Cyclohexanes; Cytochromes; Depression, Chemical; Edetic Acid; Energy Transfer; Heart; Imides; Mitochondria, Muscle; Molecular Weight; NAD; Oligomycins; Oxidation-Reduction; Oxidative Phosphorylation; Succinates
PubMed: 4299126
DOI: 10.1042/bj1080445 -
Biochemical and Biophysical Research... Apr 2014In addition to performing its essential transport function, the sodium pump also activates multiple cell signaling pathways in response to digitalis drugs such as...
In addition to performing its essential transport function, the sodium pump also activates multiple cell signaling pathways in response to digitalis drugs such as ouabain. Based mainly on cell-free studies with mixtures of purified Src kinase and Na(+)/K(+)-ATPase, a well-advocated hypothesis on how ouabain initiates the activation of signaling pathways is that there is a preexisting physiological complex of inactive Src bound to the α-subunit of Na(+)/K(+)-ATPase, and that ouabain binding to this subunit disrupts the bound Src and activates it. Because of the published disagreements of the results of such cell-free experiments of two other laboratories, our aim was to attempt the resolution of these discrepancies. We reexamined the effects of ouabain, vanadate, and oligomycin on mixtures of Src, Na(+)/K(+)-ATPase, Mg(2+), and ATP as specified in prior studies; and assayed for Src-418 autophosphorylation as the measure of Src activation. In contrast to the findings of the proponents of the above hypothesis, our results showed similar effects of the three inhibitors of Na(+)/K(+)-ATPase; indicating that Src activation in such experiments is primarily due to the ATP-sparing effect of the ATPase inhibitor on the mixture of two enzymes competing for ATP. We conclude that there is no solid evidence for direct molecular interaction of Src with Na(+)/K(+)-ATPase under physiological conditions.
Topics: Animals; Digitalis; Enzyme Inhibitors; Oligomycins; Ouabain; Phosphorylation; Protein Subunits; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Swine; Vanadates; src-Family Kinases
PubMed: 24667596
DOI: 10.1016/j.bbrc.2014.03.071 -
Biochemical and Biophysical Research... Sep 2018Platelet activation plays a key role in normal haemostasis and pathological thrombosis. Platelet activation is rapid; within minutes of stimulation, platelets generate...
Platelet activation plays a key role in normal haemostasis and pathological thrombosis. Platelet activation is rapid; within minutes of stimulation, platelets generate bioactive phospholipids, secrete their granule contents, activate integrins and aggregate together to form a haemostatic plug. These events are dependent on ATP synthesis. Mitochondrial function in platelets from healthy volunteers and patients with a range of diseases indicate an important role for oxygen consumption in oxidative phosphorylation in normal and pathological function. Platelets also consume oxygen during oxidation reactions, such as cyclooxygenase-dependent thromboxane A synthesis. In this study, we used high-resolution respirometry to investigate rapid changes in oxygen consumption during platelet activation. We demonstrated a rapid, transient increase in oxygen consumption rate within minutes of platelet stimulation by the physiological activator, thrombin. This was partly inhibited by aspirin and by oligomycin. This shows that high resolution respirometry can provide information regarding rapid and dynamic changes in oxygen consumption during platelet activation.
Topics: Aspirin; Blood Platelets; Cell Respiration; Humans; Kinetics; Oligomycins; Oxygen; Oxygen Consumption; Platelet Aggregation; Primary Cell Culture; Thrombin
PubMed: 30093113
DOI: 10.1016/j.bbrc.2018.08.031 -
The Journal of Biological Chemistry Oct 1996The mechanism whereby oligomycin occludes Na+ within Na/K-ATPase was investigated to study Na+ and K+ transport mechanisms. Oligomycin stimulated Na+ binding to...
The mechanism whereby oligomycin occludes Na+ within Na/K-ATPase was investigated to study Na+ and K+ transport mechanisms. Oligomycin stimulated Na+ binding to Na/K-ATPase but inhibited Na-K and Na-Na exchange. The oligomycin concentration required to stimulate Na+ binding to half-maximal was 4.5 microM, which was close to the concentration that reduced Na-Na and Na-K exchange and ATPase activity to half-maximal, suggesting that Na/K-ATPase possesses an oligomycin binding site responsible for stimulating Na+ binding and reducing ion exchange and ATPase activity. In contrast, neither K+ binding nor K+ transport was affected by oligomycin. Limited tryptic digestion of Na/K-ATPase showed that, unlike Na+, K+, and ouabain, oligomycin treatment did not result in a specific digestion pattern. Oligomycin appeared to inhibit ouabain binding in a noncompetitive manner, whereas it did not affect ATP binding. Na/K-ATPase isoforms with low and high sensitivities to ouabain were equally sensitive to oligomycin. These results suggest that the oligomycin binding site is located on the extracellular side of Na/K-ATPase, at a different position from the ouabain binding site, and this antibiotic did not induce a conformational change of Na/K-ATPase. We propose that oligomycin interacts with the Na+ occlusion site from the extracellular side of Na/K-ATPase, which delays Na+ release to the extracellular side without inducing a conformational change, suggesting that the pathways responsible for Na+ and K+ transport differ.
Topics: Animals; Binding Sites; Cattle; Dogs; Kidney; Kidney Medulla; Kinetics; Microsomes; Models, Structural; Oligomycins; Ouabain; Peptide Fragments; Potassium; Protein Conformation; Rubidium; Sodium; Sodium-Potassium-Exchanging ATPase
PubMed: 8810335
DOI: 10.1074/jbc.271.41.25604 -
Journal of Bacteriology Feb 1978We have compared the adenosine triphosphatase (ATPase) activity of mitochondria prepared from wild-type Neurospora crassa and from poky, a maternally inherited mutant... (Comparative Study)
Comparative Study
We have compared the adenosine triphosphatase (ATPase) activity of mitochondria prepared from wild-type Neurospora crassa and from poky, a maternally inherited mutant known to possess defective mitochondrial ribosomes and reduced amounts of cytochromes aa3 and b. poky contains two distinct forms of mitochondrial ATPase. The first is normal in its Km for ATP, specificity for nucleotides and divalent cations, pH optimum, cold stability, and sensitivity to inhibitors (oligomycin, N,N-dicyclohexyl carbodiimide, and adenylyl imidodiphosphate). The fact that membrane-bound, cold-stable, oligomycin-sensitive ATPase activity is present in poky (with an activity of 1.93 +/- 0.03 mumol/min-mg of protein compared with 1.33 +/- 0.07 mumol/min-mg of protein in the wild-type strain) and also in chloramphenicol-grown wild-type cells suggests that products of mitochondrial protein synthesis play only a limited role in the attachment of the mitochondrial ATPase to the membrane in Neurospora. poky also contains a second form of mitochondrial ATPase, which has an activity of 1.5 +/- 0.2 mumol/min-mg of protein, is oligomycin sensitive but cold labile, and presumably is attached less firmly to the mitochondrial membrane. The two forms, added together, represent a substantial overproduction of mitochondrial ATPase by poky.
Topics: Adenosine Triphosphatases; Chloramphenicol; Dicyclohexylcarbodiimide; Genotype; Hydrogen-Ion Concentration; Magnesium; Mitochondria; Mutation; Neurospora; Neurospora crassa; Oligomycins
PubMed: 24038
DOI: 10.1128/jb.133.2.584-592.1978 -
The Journal of Biological Chemistry Jan 1986In the phosphoenzyme (EP) of the electric eel Na,K-ATPase, the sum of the ADP-sensitive EP and the K+-sensitive EP exceeds 150% of EP in the presence of 100 mM Na+. This...
In the phosphoenzyme (EP) of the electric eel Na,K-ATPase, the sum of the ADP-sensitive EP and the K+-sensitive EP exceeds 150% of EP in the presence of 100 mM Na+. This unusual phenomenon can be explained by the formation of three phosphoenzymes: ADP-sensitive K+-insensitive (E1P), K+-sensitive ADP-insensitive (E2P), and ADP- and K+-sensitive (E*P) phosphoenzymes, as proposed by Nørby et al. (Nørby, J. G., Klodos, I., and Christiansen, N. O. (1983) J. Gen. Physiol. 82, 725-757). By applying a simple approximation method for the assay of E1P, E*P, and E2P, it was found that the phosphorylation of the enzyme was much faster than the conversion among each EP and the phosphoenzyme changed as E1NaATP----E1P----E*P----E2P. In the fragmental eel enzyme, the step of E*P to E2P was much slower than the step of E1P to E*P. In the steady state, the E1P was predominant above 400 mM Na+, whereas E*P and E2P were predominant between 60 and 300 mM Na+ and below 60 mM Na+, respectively. The characteristic difference of the eel enzyme from the beef brain enzyme and probably from the kidney enzyme seems to be that the dissociation constant of Na+ on the E1P-E*P equilibrium is higher than that on the E*P-E2P. The E*P and E1P both interacted with ADP to form ATP without formation of inorganic phosphate in the absence of free Mg2+. In the Na,K-ATPase proteoliposomes, the vesicle membrane interfered with the conversion of E1P to E2P, especially the change of E1P to E*P, and furthermore, the E1P content increased. This barrier effect was partially counteracted by monensin or carbonyl cyanide m-chlorophenylhydrazone. Oligomycin reacted with E1P and probably with E*P, therefore inhibiting their conversion to E2P and interaction with K+.
Topics: Adenosine Diphosphate; Animals; Electric Organ; Electrophorus; Mathematics; Oligomycins; Phosphorylation; Potassium; Proteolipids; Sodium; Sodium-Potassium-Exchanging ATPase
PubMed: 3003056
DOI: No ID Found -
The Biochemical Journal Nov 19761. When rat spleen mitochondria are incubated with oxidizable substrates, added MgCl2 (greater than 150 muM free concentration) markedly stimulates state-4 respiration...
1. When rat spleen mitochondria are incubated with oxidizable substrates, added MgCl2 (greater than 150 muM free concentration) markedly stimulates state-4 respiration and lowers both the respiratory control and ADP/O ratios; this effect is reversible on addition of excess of EDTA. 2. With [gamma-32P]ATP as substrate, an Mg2+-stimulated ATPase (adenosine triphosphate) was identified in the atractyloside-insensitive and EDTA-accessible space of intact rat spleen mitochondria. 3. Oligomycin has no effect on the activity of the Mg2+-stimulated ATPase at a concentration (2.0mug/mg of protein) that completely inhibits the atractyloside-sensitive reaction. Of the two ATPase activities, only the atracytoloside sensitive reaction is stimulated (approx. 40%) by dinitrophenol. 4. On digitonin fractionation the atractyloside-insensitive Mg2+-stimulated ATPase co-purifies with the outer membrane-fraction of rat spleen mitochondria, whereas (as expected) the atractylosidesensitive activity co-purifies with the inner-membrane plus matrix fraction. 5. Stoicheiometric amounts of ADP and Pi are produced as the end products of ATP hydrolysis by purified outer-membrane fragments; no significant AMP production is detected during the time-course of the reaction. 6. The outer-membrane ATPase is present in rat kidney cortex and heart mitochondria as well as in spleen, but is absent from rat liver, thymus, brain, lung, diaphragm and skeletal muscle.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Atractyloside; Calcium; Centrifugation, Density Gradient; Digitonin; Dinitrophenols; Electron Transport; Isoenzymes; Magnesium; Male; Mitochondria; Oligomycins; Oxygen Consumption; Rats; Spleen
PubMed: 137723
DOI: 10.1042/bj1600383