-
International Journal of Molecular... Oct 2020Herein we report the synthesis, antioxidant and neuroprotective power of homo-tris-nitrones () , designed on the hypothesis that the incorporation of a third nitrone...
Herein we report the synthesis, antioxidant and neuroprotective power of homo-tris-nitrones () , designed on the hypothesis that the incorporation of a third nitrone motif into our previously identified homo-bis-nitrone () would result in an improved and stronger neuroprotection. The neuroprotection of , measured against oligomycin A/rotenone, showed that was the best neuroprotective agent at a lower dose (EC = 51.63 ± 4.32 μM), being similar in EC and maximal activity to α-phenyl---butylnitrone () and less potent than any of . The results of neuroprotection in an in vitro oxygen glucose deprivation model showed that was the most powerful (EC = 87.57 ± 3.87 μM), at lower dose, but 50-fold higher than its analogous , and ≈1.7-fold less potent than . had a very good antinecrotic (IC = 3.47 ± 0.57 μM), antiapoptotic, and antioxidant (EC = 6.77 ± 1.35 μM) profile, very similar to that of its analogous . In spite of these results, and still being attractive neuroprotective agents, and do not have better neuroprotective properties than , but clearly exceed that of .
Topics: Antioxidants; Caspase 3; Cell Line; Cell Survival; Cyclic N-Oxides; Humans; Molecular Structure; Neurons; Neuroprotective Agents; Nitrogen Oxides; Oligomycins; Reactive Oxygen Species; Rotenone
PubMed: 33114714
DOI: 10.3390/ijms21217949 -
EMBO Reports May 2014Larvae of Drosophila melanogaster reared at 23°C and switched to 14°C for 1 h are 0.5°C warmer than the surrounding medium. In keeping with dissipation of energy,...
Larvae of Drosophila melanogaster reared at 23°C and switched to 14°C for 1 h are 0.5°C warmer than the surrounding medium. In keeping with dissipation of energy, respiration of Drosophila melanogaster larvae cannot be decreased by the F-ATPase inhibitor oligomycin or stimulated by protonophore. Silencing of Ucp4C conferred sensitivity of respiration to oligomycin and uncoupler, and prevented larva-to-adult progression at 15°C but not 23°C. Uncoupled respiration of larval mitochondria required palmitate, was dependent on Ucp4C and was inhibited by guanosine diphosphate. UCP4C is required for development through the prepupal stages at low temperatures and may be an uncoupling protein.
Topics: Animals; Cells, Cultured; Cold Temperature; Drosophila Proteins; Drosophila melanogaster; Enzyme Inhibitors; Gene Knockout Techniques; Guanosine Diphosphate; Larva; Membrane Transport Proteins; Mitochondria; Oligomycins; Oxygen Consumption; Palmitates; Respiration; Thermogenesis; Uncoupling Agents
PubMed: 24639557
DOI: 10.1002/embr.201337972 -
International Journal of Molecular... Sep 2022Platelets produce inorganic polyphosphate (polyP) upon activation to stimulate blood coagulation. Some researchers have linked polyP metabolism to ATP production,...
Platelets produce inorganic polyphosphate (polyP) upon activation to stimulate blood coagulation. Some researchers have linked polyP metabolism to ATP production, although the metabolic linkage is yet to be elucidated. We found evidence for this possibility in our previous study on professional athletes (versus non-athletes), and proposed that the regulatory mechanism might be different for these two groups. To explore this aspect further, we investigated the effects of modulated ATP production on polyP levels. Blood samples were obtained from Japanese healthy, non-athletes in the presence of acid-citrate-dextrose. The platelets in the plasma were treated with oligomycin, rotenone, and GlutaMAX to modulate ATP production. PolyP level was quantified fluorometrically and visualized using 4',6-diamidino-2-phenylindole. Correlations between polyP and ATP or NADH were then calculated. Contrary to the hypothesis, inhibitors of ATP production increased polyP levels, whereas amino acid supplementation produced the opposite effect. In general, however, polyP levels were positively correlated with ATP levels and negatively correlated with NADH levels. Since platelets are metabolically active, they exhibit high levels of ATP turnover rate. Therefore, these findings suggest that ATP may be involved in polyP production in the resting platelets of non-athletes.
Topics: Adenosine Triphosphate; Amino Acids; Citrates; Glucose; Humans; NAD; Oligomycins; Polyphosphates; Rotenone
PubMed: 36232597
DOI: 10.3390/ijms231911293 -
Cells Dec 2022Mitochondrial dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy...
Relative Importance of Different Elements of Mitochondrial Oxidative Phosphorylation in Maintaining the Barrier Integrity of Retinal Endothelial Cells: Implications for Vascular-Associated Retinal Diseases.
PURPOSE
Mitochondrial dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy and retinopathy of prematurity. Therefore, we aimed to investigate the role of different mitochondrial constituents, specifically those of oxidative phosphorylation (OxPhos), in maintaining the barrier function of RECs.
METHODS
Electric cell-substrate impedance sensing (ECIS) technology was used to assess in real time the role of different mitochondrial components in the total impedance (Z) of human RECs (HRECs) and its components: capacitance (C) and the total resistance (R). HRECs were treated with specific mitochondrial inhibitors that target different steps in OxPhos: rotenone for complex I, oligomycin for complex V (ATP synthase), and FCCP for uncoupling OxPhos. Furthermore, data were modeled to investigate the effects of these inhibitors on the three parameters that govern the total resistance of cells: Cell-cell interactions (R), cell-matrix interactions (α), and cell membrane permeability (Cm).
RESULTS
Rotenone (1 µM) produced the greatest reduction in Z, followed by FCCP (1 µM), whereas no reduction in Z was observed after oligomycin (1 µM) treatment. We then further deconvoluted the effects of these inhibitors on the R, α, and C parameters. Rotenone (1 µM) completely abolished the resistance contribution of R, as the R became zero immediately after the treatment. Secondly, FCCP (1 µM) eliminated the resistance contribution of R only after 2.5 h and increased C without a significant effect on α. Lastly, of all the inhibitors used, oligomycin had the lowest impact on R, as evidenced by the fact that this value became similar to that of the control group at the end of the experiment without noticeable effects on C or α.
CONCLUSION
Our study demonstrates the differential roles of complex I, complex V, and OxPhos coupling in maintaining the barrier functionality of HRECs. We specifically showed that complex I is the most important component in regulating HREC barrier integrity. These observed differences are significant since they could serve as the basis for future pharmacological and gene expression studies aiming to improve the activity of complex I and thereby provide avenues for therapeutic modalities in endothelial-associated retinal diseases.
Topics: Infant, Newborn; Humans; Oxidative Phosphorylation; Rotenone; Endothelial Cells; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Mitochondria; Diabetic Retinopathy; Oligomycins
PubMed: 36552890
DOI: 10.3390/cells11244128 -
The Journal of Physiology Sep 19671. When resealed ghosts containing adenosine triphosphate (ATP), magnesium and sodium were incubated in a medium containing potassium, ATP was hydrolysed vigorously by a...
1. When resealed ghosts containing adenosine triphosphate (ATP), magnesium and sodium were incubated in a medium containing potassium, ATP was hydrolysed vigorously by a ouabain-sensitive mechanism. If the ghosts contained potassium instead of or in addition to sodium, and the external solution contained sodium but no potassium, there was little ouabain-sensitive hydrolysis of ATP. As it is known that the ouabain-sensitive ATPase in fragmented ghosts requires both sodium and potassium ions, these results show that the ATPase is activated by potassium externally and by sodium internally, and suggest that the ions activating the ATPase are the ions that are transported.2. Resealed ghosts containing ATP, magnesium and sodium were incubated in sodium-free media containing potassium, with and without ouabain, and the rate of loss of sodium and rate of hydrolysis of ATP were measured. The hydrolysis of 1 molecule of ATP by the ouabain-sensitive mechanism was accompanied by the ouabain-sensitive loss of about 3 sodium ions.3. (24)Na and (42)K were used to measure sodium efflux and potassium influx in identical batches of fresh red cells under the same conditions and at the same time. Each flux was measured in the presence and absence of ouabain. The ratio (ouabain-sensitive sodium efflux)/(ouabain-sensitive potassium influx) was significantly greater than 1 (1.20 +/- 0.01 and 1.35 +/- 0.01 in two experiments). If a small fraction of the potassium influx represented a ouabain-sensitive potassium: potassium exchange, the ratio of the numbers of ions moved in the sodium: potassium exchange catalysed by the pump must have been even further from unity.4. Resealed ghosts containing [gamma-(32)P]ATP, magnesium, (24)Na and orthophosphate were incubated in balanced salt solutions with and without potassium and with and without ouabain. A comparison of sodium efflux, estimated from (24)Na loss, with ATP hydrolysis, estimated from the formation of [(32)P]orthophosphate, showed that the sodium:sodium exchange in a potassium-free medium was accompanied by little or no ouabain-sensitive hydrolysis of ATP.5. Experiments on intact red cells loaded with (24)Na showed that both sodium:sodium exchange in a potassium-free medium, and sodium:potassium exchange in a medium containing potassium, were partially inhibited by oligomycin (1-10 mug/ml.). Inhibition of the sodium:potassium exchange was not affected by raising the external potassium concentration.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Biological Transport; Cell Membrane; Cell Membrane Permeability; Erythrocytes; Oligomycins; Ouabain; Potassium; Sodium
PubMed: 4228075
DOI: 10.1113/jphysiol.1967.sp008297 -
Scientific Reports May 2021Direct and real-time monitoring of cerebral metabolism exploiting the drastic increase in sensitivity of hyperpolarized C-labeled metabolites holds the potential to...
Direct and real-time monitoring of cerebral metabolism exploiting the drastic increase in sensitivity of hyperpolarized C-labeled metabolites holds the potential to report on neural activity via in-cell metabolic indicators. Here, we followed the metabolic consequences of curbing action potential generation and ATP-synthase in rat cerebrum slices, induced by tetrodotoxin and oligomycin, respectively. The results suggest that pyruvate dehydrogenase (PDH) activity in the cerebrum is 4.4-fold higher when neuronal firing is unperturbed. The PDH activity was 7.4-fold reduced in the presence of oligomycin, and served as a pharmacological control for testing the ability to determine changes to PDH activity in viable cerebrum slices. These findings may open a path towards utilization of PDH activity, observed by magnetic resonance of hyperpolarized C-labeled pyruvate, as a reporter of neural activity.
Topics: Action Potentials; Adenosine Triphosphate; Animals; Brain; Cerebrum; Female; Magnetic Resonance Spectroscopy; Mitochondrial Proton-Translocating ATPases; Oligomycins; Oxidation-Reduction; Oxidoreductases; Pyruvate Dehydrogenase Complex; Pyruvic Acid; Rats; Rats, Sprague-Dawley; Tetrodotoxin
PubMed: 33986346
DOI: 10.1038/s41598-021-89534-4 -
Scientific Reports Oct 2021Intestinal epithelial cells (IECs) are crucial to maintain intestinal function and the barrier against the outside world. To support their function they rely on energy...
Intestinal epithelial cells (IECs) are crucial to maintain intestinal function and the barrier against the outside world. To support their function they rely on energy production, and failure to produce enough energy can lead to IEC malfunction and thus decrease intestinal barrier function. However, IEC metabolic function is not often used as an outcome parameter in intervention studies, perhaps because of the lack of available methods. We therefore developed a method to isolate viable IECs, suitable to faithfully measure their metabolic function by determining extracellular glycolytic and mitochondrial flux. First, various methods were assessed to obtain viable IECs. We then adapted a previously in-house generated image-analysis algorithm to quantify the amount of seeded IECs. Correcting basal respiration data of a group of piglets using this algorithm reduced the variation, showing that this algorithm allows for more accurate analysis of metabolic function. We found that delay in metabolic analysis after IEC isolation decreases their metabolic function and should therefore be prevented. The presence of antibiotics during isolation and metabolic assessment also decreased the metabolic function of IECs. Finally, we found that primary pig IECs did not respond to Oligomycin, a drug that inhibits complex V of the electron transport chain, which may be because of the presence of drug exporters. A method was established to faithfully measure extracellular glycolytic and mitochondrial flux of pig primary IECs. This tool is suitable to gain a better understanding of how interventions affect IEC metabolic function.
Topics: Animals; Anti-Bacterial Agents; Epithelial Cells; Extracellular Fluid; Glycolysis; Intestinal Mucosa; Mitochondria; Oligomycins; Swine
PubMed: 34620944
DOI: 10.1038/s41598-021-99460-0 -
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 -
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 -
Biochimica Et Biophysica Acta.... Sep 2018Enzymes in the respiratory chain are increasingly seen as potential targets against multi-drug resistance of human pathogens and cancerous cells. However, a detailed...
Enzymes in the respiratory chain are increasingly seen as potential targets against multi-drug resistance of human pathogens and cancerous cells. However, a detailed understanding of the mechanism and specificity determinants of known inhibitors is still lacking. Oligomycin, for example, has been known to be an inhibitor of the membrane motor of the mitochondrial ATP synthase for over five decades, and yet little is known about its mode of action at the molecular level. In a recent breakthrough, a crystal structure of the S. cerevisiae c-subunit ring with bound oligomycin revealed the inhibitor docked on the outer face of the proton-binding sites, deep into the transmembrane region. However, the structure of the complex was obtained in an organic solvent rather than detergent or a lipid bilayer, and therefore it has been unclear whether this mode of recognition is physiologically relevant. Here, we use molecular dynamics simulations to address this question and gain insights into the mechanism of oligomycin inhibition. Our findings lead us to propose that oligomycin naturally partitions into the lipid/water interface, and that in this environment the inhibitor can indeed bind to any of the c-ring proton-carrying sites that are exposed to the membrane, thereby becoming an integral component of the proton-coordinating network. As the c-ring rotates within the membrane, driven either by downhill proton permeation or ATP hydrolysis, one of the protonated, oligomycin-bound sites eventually reaches the subunit-a interface and halts the rotary mechanism of the enzyme.
Topics: Adenosine Triphosphate; Binding Sites; Enzyme Inhibitors; Mitochondrial Membranes; Mitochondrial Proton-Translocating ATPases; Molecular Dynamics Simulation; Oligomycins; Protein Conformation; Saccharomyces cerevisiae
PubMed: 29630891
DOI: 10.1016/j.bbabio.2018.03.019