-
The Biochemical Journal Oct 1975The uptake and the washout of 45Ca2+ and 32Pi is described in free fat-cells and whole epididymal fat-pads from fed rats. 2. In isolated fat-cells, the uptake of 45Ca2+...
The uptake and the washout of 45Ca2+ and 32Pi is described in free fat-cells and whole epididymal fat-pads from fed rats. 2. In isolated fat-cells, the uptake of 45Ca2+ proceeds with an initial rapid phase of about 1 min duration, followed by a slower subsequent accumulation. In contrast with the rapid phase, the slow phase is inhibited by 2,4-dinitrophenol, warfarin, oligomycin and verapamil, shows saturation, and presumably represents transport across the plasma membrane. 3. The washout of 45Ca2+ from preloaded cells consists of a rapid (1 min) initial phase and a slow phase which is non-monoexponential, suggesting that the radioactive isotope is released from several cellular pools. 4. When Pi is omitted from the incubation medium, the slow phase of 45Ca uptake is almost abolished, and the washout of 45Ca from preloaded fat-cells is markedly accelerated. At elevated extracellular concentrations of Pi (2,4-6.2mM), the uptake of 45Ca is stimulated by 2-10-fold, and the release of the radioactive isotope from preloaded cells is inhibited. In whole epididymal fat-pads, variations in the extracellular concentration of Pi have no detectable effect on the uptake or the washout of 45Ca. 5. In isolated fat-cells, the accumulation of 32Pi is inhibited by 2,4-dinitrophenol or the omission of glucose from the incubation medium. In a Ca2+-depleted buffer, the uptake of 32Pi is diminished, and hyperosmolarity, which stimulates 45Ca uptake, also accelerates the accumulation of 32Pi. 6. It is concluded that in free fat-cells, the uptake and release of Ca2+ and Pi take place by closely interrelated processes, which are dependent on mitochondrial energy production.
Topics: Adipose Tissue; Animals; Biological Transport, Active; Calcium; Cell Separation; Dinitrophenols; Glucose; In Vitro Techniques; Male; Oligomycins; Phosphates; Rats; Time Factors; Verapamil; Warfarin
PubMed: 1212220
DOI: 10.1042/bj1520121 -
European Journal of Biochemistry Jun 1975The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of "sonic" submitochondrial particles has been...
The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of "sonic" submitochondrial particles has been studied. 1. Octylguanidine inhibits state 3 respiration with glutamate-malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine. 2. The titration-curve for the octylguanidine inhibition of glutamate-malate oxidation is hyperbolic and apparently biphasic, half-maximal inhibition is obtained at 30 muM octylguanidine. The octylguanidine-curve for inhibition of succinate oxidation is sigmoid with half-maximal inhibition at about 250 muM. 3. Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malage and succinate as respiratory substrates. 4. Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together. 5. Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration-curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive. 6. It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site I phosphorylation to octylguanidine.
Topics: Animals; Guanidines; In Vitro Techniques; Kinetics; Mitochondria, Liver; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Rats
PubMed: 1175599
DOI: 10.1111/j.1432-1033.1975.tb02131.x -
Genetics Jul 1975A meiotic segregant (oliPR1) was isolated with a phenotype of multiple cross resistance and collateral sensitivity. Strain oliPR1 has increased sensitivity to ethidium...
A meiotic segregant (oliPR1) was isolated with a phenotype of multiple cross resistance and collateral sensitivity. Strain oliPR1 has increased sensitivity to ethidium bromide, dequalinium chloride, acriflavin, paromomycin and neomycin, and increased resistance to oligomycin, rutamycin, venturicidin, triethyltin bromide, antimycin, carbonylcynamide-m-chlorophenylhydrazone, tetra-N-butylammonium bromide, dibenzyldimethylammonium chloride, triphenylmethlphosphonium bromide, chloramphenicol, carbomycin, tetracycline, triton X-165 and cycloheximide. Single gene inheritance of the cross resistance and collateral sensitivity was shown by 2:2 parental ditype segregation and reversion of the complete phenotype by a spontaneous revertant. The locus conferring the oliPR1 phenotype was mapped 11.7 units from an unspecified centromere. Antibiotic resistance showed incomplete dominance, with the level of hybrid resistance dependent upon the inhibitor tested. Resistant diploids that produced four resistant ascospores were the result of mitotic recombination prior to meiosis. A partial revertant phenotype (sensitive to all inhibitors except oligomycin, antimycin and carbonylcyanide-m-chlorophenylhydrazone) was shown to be due to a single nuclear gene causing partial suppression of oliPR1. Anaerobic pretreatment, 37degrees and 0.5 MKC1 were observed to reduce the growth of oliPR1 when challenged with seven diverse inhibitors (antimycin, carbonylcyanide-m-chlorophenylhydrazone,-chloramphenicol, cycloheximide, oligomycin, triethyltin bromide, and triphenylmethylphosphonium bromide). Resistance to cycloheximide was not altered by the [rho--] state. A revertant of oliPR1 (sensitive to the above inhibitors but resistant to ethidium bromide, paromycin and neomycin) showed anaerobic and temperature sensitization to ethidium bromide, paromomycin and neomycin. Continuous monitoring of oxygen uptake by the revertant afteranaerobic pretreatment revealed that anaerbiosis sensitized respiratory adaptation of the revertant to neomycin. It is proposed that oliPR1 is a mutation resulting in the alteration of plasma membrane permeability to many diverse inhibitors.
Topics: Antimycin A; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Chloramphenicol; Cycloheximide; Drug Resistance, Microbial; Erythromycin; Ethidium; Genes; Mutation; Neomycin; Oligomycins; Phenotype; Saccharomyces cerevisiae; Terphenyl Compounds; Tetracycline; Triethyltin Compounds
PubMed: 782999
DOI: No ID Found -
The Biochemical Journal Jun 1997Inhibitors of mitochondrial oxidative metabolism have been proposed to interfere with Ca2+ influx mediated by store-operated channels (SOC), secondary to their effects...
Inhibitors of mitochondrial oxidative metabolism have been proposed to interfere with Ca2+ influx mediated by store-operated channels (SOC), secondary to their effects on ATP production. We assessed SOC activity by 45Ca2+ influx and fluorimetric measurements of free Ca2+ or Mn2+ quench in thapsigargin-treated Chinese hamster ovary cells and Jurkat T-cells, and additionally by electrophysiological measurements of the Ca2+-release-activated Ca2+ current (Icrac) in Jurkat T-cells. Various mitochondrial antagonists were confirmed to inhibit SOC. However, the following evidence supported the proposal that oligomycin, in particular, exerts an inhibitory effect on SOC in addition to its known actions on mitochondria and Na+-pump activity: (i) the concentrations of oligomycin required to inhibit SOC-mediated Ca2+ influx or Icrac (half-inhibitory concentration approximately 2 microM) were nearly 50-fold higher than the concentrations that blocked mitochondrial ATP production; (ii) the rank order of potency of oligomycins A, B and C for decreasing SOC-mediated Ca2+ influx or Icrac differed from that known for inhibition of mitochondrial function; (iii) oligomycin blocked Icrac under voltage clamp and with intracellular Na+ and K+ concentrations fixed by dialysis from the patch pipette, arguing that the effect was not secondary to membrane polarization or pump activity; and (iv) fixing the cytosolic ATP concentration by dialysis from the patch pipette attenuated rotenone- but not oligomycin-mediated inhibition of Icrac. Oligomycin also blocked volume-activated Cl- currents, a profile common to some other known blockers of SOC that are not known mitochondrial inhibitors. These findings raise the possibility that oligomycin interacts directly with SOC, and thus may extend the known pharmacological profile for this type of Ca2+-influx pathway.
Topics: Adenosine Triphosphate; Animals; CHO Cells; Calcium; Cricetinae; Humans; Ion Channels; Ion Transport; Jurkat Cells; Manganese; Mitochondria; Oligomycins
PubMed: 9210424
DOI: 10.1042/bj3240971 -
British Journal of Haematology Apr 2014Drug-resistant forms of acute lymphoblastic leukaemia (ALL) are a leading cause of death from disease in children. Up to 25% of patients with T-cell ALL (T-ALL) develop...
Drug-resistant forms of acute lymphoblastic leukaemia (ALL) are a leading cause of death from disease in children. Up to 25% of patients with T-cell ALL (T-ALL) develop resistance to chemotherapeutic agents, particularly to glucocorticoids (GCs), a class of drug to which resistance is one of the strongest indicators of poor clinical outcome. Despite their clinical importance, the molecular mechanisms that underpin GC resistance and leukaemia relapse are not well understood. Recently, we demonstrated that GC-resistance is associated with a proliferative metabolism involving the up-regulation of glycolysis, oxidative phosphorylation and cholesterol biosynthesis. Here we confirm that resistance is directly associated with a glycolytic phenotype and show that GC-resistant T-ALL cells are able to shift between glucose bioenergetic pathways. We evaluated the potential for targeting these pathways in vitro using a glycolysis inhibitor, 2-deoxyglucose (2DG), and the oxidative phosphorylation inhibitor oligomycin in combination with methylprednisolone (MPRED). We found that oligomycin synergized with MPRED to sensitize cells otherwise resistant to GCs. Similarly we observed synergy between MPRED and simvastatin, an inhibitor of cholesterol metabolism. Collectively, our findings suggest that dual targeting of bioenergetic pathways in combination with GCs may offer a promising therapeutic strategy to overcome drug resistance in ALL.
Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; Galactose; Glucocorticoids; Glycolysis; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Methylprednisolone; Mitochondria; Oligomycins; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Signal Transduction
PubMed: 24456076
DOI: 10.1111/bjh.12727 -
PloS One 2015The biological consequences upon exposure of cells in culture to a dose of xenobiotic are not only dependent on biological variables, but also the physical aspects of...
BACKGROUND
The biological consequences upon exposure of cells in culture to a dose of xenobiotic are not only dependent on biological variables, but also the physical aspects of experiments e.g. cell number and media volume. Dependence on physical aspects is often overlooked due to the unrecognized ambiguity in the dominant metric used to express exposure, i.e. initial concentration of xenobiotic delivered to the culture medium over the cells. We hypothesize that for many xenobiotics, specifying dose as moles per cell will reduce this ambiguity. Dose as moles per cell can also provide additional information not easily obtainable with traditional dosing metrics.
METHODS
Here, 1,4-benzoquinone and oligomycin A are used as model compounds to investigate moles per cell as an informative dosing metric. Mechanistic insight into reactions with intracellular molecules, differences between sequential and bolus addition of xenobiotic and the influence of cell volume and protein content on toxicity are also investigated.
RESULTS
When the dose of 1,4-benzoquinone or oligomycin A was specified as moles per cell, toxicity was independent of the physical conditions used (number of cells, volume of medium). When using moles per cell as a dose-metric, direct quantitative comparisons can be made between biochemical or biological endpoints and the dose of xenobiotic applied. For example, the toxicity of 1,4-benzoquinone correlated inversely with intracellular volume for all five cell lines exposed (C6, MDA-MB231, A549, MIA PaCa-2, and HepG2).
CONCLUSIONS
Moles per cell is a useful and informative dosing metric in cell culture. This dosing metric is a scalable parameter that: can reduce ambiguity between experiments having different physical conditions; provides additional mechanistic information; allows direct comparison between different cells; affords a more uniform platform for experimental design; addresses the important issue of repeatability of experimental results, and could increase the translatability of information gained from in vitro experiments.
Topics: Benzoquinones; Cell Count; Cell Line; Cell Size; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Glutathione; Hep G2 Cells; Humans; Models, Biological; Oligomycins; Osmolar Concentration; Proteins; Reproducibility of Results; Xenobiotics
PubMed: 26172833
DOI: 10.1371/journal.pone.0132572 -
The Journal of Biological Chemistry Sep 1984We present experiments designed to test whether or not the ADP-sensitive (E1P) and the K-sensitive (E2P) phosphoenzyme forms of (Na,K)-ATPase are intermediates of ATP...
We present experiments designed to test whether or not the ADP-sensitive (E1P) and the K-sensitive (E2P) phosphoenzyme forms of (Na,K)-ATPase are intermediates of ATP hydrolysis in the presence of Na and K. We have used the apparent rate constant of anthroylouabain binding, k' on, and its inhibition by oligomycin to monitor the steady-state levels of E2P and E1P, respectively. We have measured k' on in purified dog kidney (Na,K)-ATPase at 37 degrees C as a function of Na, K, and MgATP or Mg plus inorganic phosphate in the presence and absence of oligomycin. In the presence of 35 mM Na and no K, k' on was saturated with [ATP] greater than 25 microM, and oligomycin (10 microM) inhibited less than 10%. Inhibition by oligomycin increased when [Na] was increased. At 1.9 M Na oligomycin inhibited k' on and equilibrium binding of anthroylouabain with Ki = 136 nM. Oligomycin had no effect on k' on in the presence of Mg + inorganic phosphate. In the presence of 35 mM Na and 4.5 mM K, k' on was much slower, inhibition by oligomycin increased, and ATP stimulated both k' on and inhibition by oligomycin with K0.5 greater than 0.1 mM. From the k' on measurements in the presence and absence of K, we estimated that the steady-state level of E2P was between 4 and 15% of the enzyme with 25 microM to 6.5 mM ATP, 35 mM Na, and 4.5 mM K at 37 degrees C. For these conditions we estimated the rate constant of E2P hydrolysis to be 46,000 (+/- 1,000) min-1. These results are contrary to the predictions derived from a model in which acid-stable phosphoenzymes are not intermediates of ATP hydrolysis in the presence of Na and K. They are fully consistent with a model in which E1P and E2P are intermediates of ATP hydrolysis coupled to transport of Na and K.
Topics: Adenosine Triphosphate; Animals; Anthracenes; Dogs; Fluorescent Dyes; Kidney; Kinetics; Mathematics; Models, Chemical; Oligomycins; Ouabain; Potassium; Sodium-Potassium-Exchanging ATPase
PubMed: 6088533
DOI: No ID Found -
European Journal of Biochemistry Jul 2000We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the...
F1 and F0 connections in the bovine mitochondrial ATP synthase: the role of the of alpha subunit N-terminus, oligomycin-sensitivity conferring protein (OCSP) and subunit d.
We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated. The results obtained show that the alpha subunit N-terminus is essential for the correct, functional connection of F1 to F0. The alpha-subunit N-terminus contacts OSCP which, in turn, contacts the F0I-PVP(b) and the F0-d subunits. The N-terminus of subunit alpha, OSCP, a segment of subunit d and the C-terminal and central region of F0I-PVP(b) subunits are peripherally located with respect to subunits gamma and delta which are completely shielded in the F1F0 complex against trypsin digestion. This qualifies the N-terminus of subunit alpha, OSCP, subunit d and F0I-PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the gamma subunit uncovered, surround the gamma and the delta subunits located in the central stalk.
Topics: Adenosine Triphosphatases; Animals; Carrier Proteins; Cattle; Cross-Linking Reagents; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Immunoblotting; Intracellular Membranes; Kinetics; Light; Liposomes; Membrane Proteins; Mitochondria; Mitochondrial Proton-Translocating ATPases; Oligomycins; Protein Structure, Tertiary; Proton-Translocating ATPases; Protons; Time Factors; Trypsin
PubMed: 10880968
DOI: 10.1046/j.1432-1327.2000.01492.x -
MBio Feb 2022In this study, 18 predicted membrane-localized ABC transporters of Candida glabrata were deleted individually to create a minilibrary of knockouts (KO). The transporter...
In this study, 18 predicted membrane-localized ABC transporters of Candida glabrata were deleted individually to create a minilibrary of knockouts (KO). The transporter KOs were analyzed for their susceptibility toward antimycotic drugs. Although Cg has previously been reported to be upregulated in various azole-resistant clinical isolates of C. glabrata, deletion of this gene did not change the susceptibility to any of the tested azoles. Additionally, CgΔ showed no change in susceptibility toward oligomycin, which is otherwise a well-known substrate of Yor1 in other yeasts. The role of CgYor1 in azole susceptibility only became evident when the major transporter Cg gene was deleted. However, under nitrogen-depleted conditions, CgΔ demonstrated an azole-susceptible phenotype, independent of CgCdr1. Notably, Cg cells also showed increased susceptibility to target of rapamycin (TOR) and calcineurin inhibitors. Moreover, increased phytoceramide levels in CgΔ and the deletions of regulators downstream of TOR and the calcineurin signaling cascade (CgΔ, Cg, CgΔ, and CgΔ) in the CgΔ background and their associated fluconazole (FLC) susceptibility phenotypes confirmed their involvement. Collectively, our findings show that TOR and calcineurin signaling govern CgYor1-mediated azole susceptibility in C. glabrata. The increasing incidence of Candida glabrata infections in the last 40 years is a serious concern worldwide. These infections are usually associated with intrinsic azole resistance and increasing echinocandin resistance. Efflux pumps, especially ABC transporter upregulation, are one of the prominent mechanisms of azole resistance; however, only a few of them are characterized. In this study, we analyzed the mechanisms of azole resistance due to a multidrug resistance-associated protein (MRP) subfamily ABC transporter, CgYor1. We demonstrate for the first time that CgYor1 does not transport oligomycin but is involved in azole resistance. Under normal growing conditions its function is masked by major transporter CgCdr1; however, under nitrogen-depleted conditions, it displays its azole resistance function independently. Moreover, we propose that the azole susceptibility due to removal of CgYor1 is not due to its transport function but involves modulation of TOR and calcineurin cascades.
Topics: Antifungal Agents; ATP-Binding Cassette Transporters; Azoles; Calcineurin; Candida glabrata; Candidiasis; Drug Resistance, Fungal; Fluconazole; Membrane Transport Proteins; Microbial Sensitivity Tests; Nitrogen; Oligomycins; Sirolimus; Fungal Proteins
PubMed: 35038899
DOI: 10.1128/mbio.03545-21 -
International Journal of Molecular... Oct 2022Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in...
Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1β was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1β, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1β). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1β (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.
Topics: Toll-Like Receptor 5; Allergens; Interleukin-10; Flagellin; Hexokinase; Glutaminase; Ligands; Antimycin A; Cerulenin; Dendritic Cells; Recombinant Proteins; Cytokines; Adjuvants, Immunologic; Vaccines; Recombinant Fusion Proteins; Glycolysis; TOR Serine-Threonine Kinases; Deoxyglucose; Oligomycins; Fatty Acids
PubMed: 36293550
DOI: 10.3390/ijms232012695