oligomycins and antimycin

In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD

Topics: ADP-Ribosylation; Animals; Antimycin A; Cell Line; Cell Line, Tumor; Cell Nucleus; Chromatin; Electron Transport; HeLa Cells; Humans; Hydrogen Peroxide; Methacrylates; Mice; Mice, Inbred C57BL; Mitochondria; Myoblasts; NAD; Oligomycins; Osteoblasts; Poly (ADP-Ribose) Polymerase-1; Rotenone; Thiazoles

In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including

Topics: 3-Hydroxyacyl CoA Dehydrogenases; Activating Transcription Factor 4; Adaptor Proteins, Signal Transducing; Amino Acids; Antimycin A; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Culture Media; eIF-2 Kinase; Gene Editing; Gene Expression Regulation; Genome, Human; Glucose; HEK293 Cells; Humans; Insulin Receptor Substrate Proteins; Mechanistic Target of Rapamycin Complex 1; Mitochondria; Neoplasm Proteins; Oligomycins; Protein Serine-Threonine Kinases; RNA, Guide, Kinetoplastida; Serine-Arginine Splicing Factors; Signal Transduction

Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP.

Topics: Adenosine Triphosphate; Animals; Anti-Bacterial Agents; Antimycin A; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cells, Cultured; Female; Granulosa Cells; Mitochondria; Oligomycins; Oocytes; Proton Ionophores; Reactive Oxygen Species; Swine; Uncoupling Agents

Mitochondrial DNA damage, increased production of reactive oxygen species and progressive respiratory chain dysfunction, together with increased deposition of cholesterol and cholesteryl esters, are hallmarks of atherosclerosis. This study investigated the role of mitochondrial function in regulation of macrophage cholesterol efflux to apolipoprotein A-I, by the addition of established pharmacological modulators of mitochondrial function.. Murine RAW 264.7 macrophages were treated with a range of concentrations of resveratrol, antimycin, dinitrophenol, nigericin and oligomycin, and changes in viability, cytotoxicity, membrane potential and ATP, compared with efflux of [3H]cholesterol to apolipoprotein (apo) A-I. The effect of oligomycin treatment on expression of genes implicated in macrophage cholesterol homeostasis were determined by quantitative polymerase chain reaction, and immunoblotting, relative to the housekeeping enzyme, Gapdh, and combined with studies of this molecule on cholesterol esterification, de novo lipid biosynthesis, and induction of apoptosis. Significant differences were determined using analysis of variance, and Dunnett's or Bonferroni post t-tests, as appropriate.. The positive control, resveratrol (24 h), significantly enhanced cholesterol efflux to apoA-I at concentrations ≥30 μM. By contrast, cholesterol efflux to apoA-I was significantly inhibited by nigericin (45%; p<0.01) and oligomycin (55%; p<0.01), under conditions (10 μM, 3 h) which did not induce cellular toxicity or deplete total cellular ATP content. Levels of ATP binding cassette transporter A1 (ABCA1) protein were repressed by oligomycin under optimal efflux conditions, despite paradoxical increases in Abca1 mRNA. Oligomycin treatment did not affect cholesterol biosynthesis, but significantly inhibited cholesterol esterification following exposure to acetylated LDL, and induced apoptosis at ≥30 μM. Finally, oligomycin induced the expression of genes implicated in both cholesterol efflux (Abca1, Abcg4, Stard1) and cholesterol biosynthesis (Hmgr, Mvk, Scap, Srebf2), indicating profound dysregulation of cholesterol homeostasis.. Acute loss of mitochondrial function, and in particular Δψm, reduces cholesterol efflux to apoA-I and dysregulates macrophage cholesterol homeostasis mechanisms. Bioavailable antioxidants, targeted to mitochondria and capable of sustaining effective mitochondrial function, may therefore prove effective in maintenance of arterial health.

Topics: Animals; Antimycin A; Apolipoproteins A; Atherosclerosis; Cell Line; Cell Survival; Cholesterol; Cholesterol Esters; DNA, Mitochondrial; Macrophages; Membrane Potential, Mitochondrial; Mice; Mitochondria; Nigericin; Oligomycins; Reactive Oxygen Species; Resveratrol; RNA, Messenger; Stilbenes

Activation of the LH receptor (LHR) in Leydig cells results in the phosphorylation of ERK1/2 by cAMP-dependent and cAMP-independent pathways. Here we examine the mechanisms by which cAMP stimulates ERK1/2 phosphorylation. We show that the stimulation of steroidogenesis is not necessary or sufficient to stimulate the phosphorylation of ERK1/2 but that other cAMP-dependent mitochondrial functions are involved. Using MA-10 cells as a model, we showed that cAMP analogs increase reactive oxygen species (ROS) formation and that an uncoupler of oxidative phosphorylation and a ROS scavenger prevent this increase. These two compounds also inhibit the increase in ERK1/2 phosphorylation provoked by cAMP analogs, thus suggesting that the cAMP-induced phosphorylation of ERK1/2 is mediated by mitochondrial ROS. In agreement with this hypothesis we also show that a reduction in glutathione levels, which alters the redox state of MA-10 cells, potentiates the effect of cAMP on ERK1/2 phosphorylation. Measurements of the dephosphorylation of ERK and the activation of Ras showed that the ROS scavenger prevents the cAMP-provoked activation of Ras and that cAMP, with or without a ROS scavenger, has little or no effect on the dephosphorylation of ERK. Lastly, we show that the uncoupler of oxidative phosphorylation and the ROS scavenger also prevent the ability of cAMP analogs to increase ERK1/2 phosphorylation in primary cultures of mouse Leydig cells. We conclude that, in Leydig cells, cAMP enhances the phosphorylation of ERK1/2 via a mitochondria-derived, ROS-dependent activation of Ras.

Topics: Acetylcysteine; Animals; Antimycin A; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Chorionic Gonadotropin; Cyclic AMP; Cyclic CMP; Enzyme Activation; Glutathione; Leydig Cells; Male; Mice; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oligomycins; Phosphorylation; Progesterone; ras Proteins; Reactive Oxygen Species; Rotenone; Uncoupling Agents

Mitochondrial dysfunction induces apoptosis of pancreatic β-cells and leads to type 2 diabetes, but the mechanism involved in this process remains unclear. Chronic endoplasmic reticulum (ER) stress plays a role in the apoptosis of pancreatic β-cells; therefore, in current study, we investigated the implication of ER stress in mitochondrial dysfunction-induced β-cells apoptosis. Metabolic stress induced by antimycin or oligomycin was used to impair mitochondrial function in MIN6N8 cells, which are mouse pancreatic β-cells. Impaired mitochondria dysfunction increased ER stress proteins such as p-eIF2α, GRP78 and GRP 94, as well as ER stress-associated apoptotic factor, CHOP, and activated JNK. AMP-activated protein kinase (AMPK) was also activated under mitochondria dysfunction by metabolic stress. However, the inhibition of AMPK by treatment with compound C, inhibitor of AMPK, and overexpression of mutant dominant negative AMPK (AMPKK45R) blocked the induction of ER stress, which was consist-ent with the decreased β-cell apoptosis and increase of insulin content. Furthermore, mitochondrial dysfunction increased the expression of the inducible nitric oxide synthase (iNOS) gene and the production of nitric oxide (NO), but NO production was prevented by compound C and mutant dominant negative AMPK (AMPK-K45R). Moreover, treatment with 1400W, which is an inhibitor of iNOS, prevented ER stress and apoptosis induced by mitochondrial dysfunction. Treatment of MIN6N8 cells with lipid mixture, physiological conditions of impaired mitochondria function, activated AMPK, increased NO production and induced ER stress. Collectively, these data demonstrate that mitochondrial dysfunction activates AMPK, which induces ER stress via NO production, resulting in pancreatic β-cells apoptosis.

Topics: AMP-Activated Protein Kinases; Animals; Antimycin A; Apoptosis; Cell Line; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Insulin; Insulin-Secreting Cells; MAP Kinase Kinase 4; Membrane Glycoproteins; Mice; Mitochondria; Nitric Oxide; Nitric Oxide Synthase; Oligomycins; Stress, Physiological; Transcription Factor CHOP

Bleomycin is a highly potent cytotoxic and genotoxic agent used in the chemotherapy of various types of tumors. It is a radiomimetic anticancer drug that produces single- and double-stranded DNA breaks in a catalytic way. Using Saccharomyces cerevisiae as a model system, we show that when a high amount of bleomycin molecules is internalized (100 micromol/L), morphological changes identical to those usually associated with apoptosis, i.e., a sub-G1 region peak, chromatin condensation, and very rapid DNA fragmentation into oligonucleosomal-sized fragments, are observed. The known bleomycin inhibitors cobalt and EDTA were able to prevent bleomycin nucleasic activity and thus apoptotic cell death. However, both oligomycin, a potent inhibitor of the mitochondrial F0F1-ATPase, and antimycin, a drug affecting mitochondria respiration, were unable to prevent the bleomycin-induced apoptotic-like cell death. These results suggest that high bleomycin concentrations induce an apoptosis-like mitochondria-independent cell death in yeast.

Topics: Antibiotics, Antineoplastic; Antimycin A; Apoptosis; Bleomycin; Chromatin Assembly and Disassembly; DNA Fragmentation; Edetic Acid; Mitochondria; Oligomycins; Saccharomyces cerevisiae

A fourth intracellular Ca2+ pool in Leishmania donovani was identified by permeabilizing plasma membrane with digitonin. In Fura 2 loaded cells Ca2+ was released synergistically when mitochondrial function was blocked by antimycin and oligomycin. Vanadate did not have any effect if applied before incorporation of these mitochondrial poisons. However, the same inhibitor which inhibits Ca2+-ATPase activity of endoplasmic reticulum was able to release Ca2+ at a slow rate when added after antimycin and oligomycin. Alkalization of cytoplasmic pH allowed further release of Ca2+ essentially from the acidocalcisome. Purified glycosomes could mediate Ca2+ uptake mechanism in presence of vanadate whereas bafilomycin, a specific and potent inhibitor of vacuolar proton pump did not have any effect. Glycosomal Ca2+-ATPase activity was optimum at pH 7.5. The apparent Km for calciumin presence of vanadate was 12 nM. Taken together, it may be suggested that a vanadate-insensitive Ca2+-ATPase is present in the membrane of this microbody. Presence of glycosomal Ca2+ was further confirmed by imaging of Ca2+ activity in the Fura 2 loaded purified organelle using confocal laser. Results reveal that newly localized glycosomal calcium may essentially be an effective candidate to play a significant role in cellular function.

Topics: Animals; Antimycin A; Calcimycin; Calcium; Calcium-Transporting ATPases; Digitonin; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Ionophores; Leishmania donovani; Macrolides; Microbodies; Microscopy, Confocal; Oligomycins; Spectrometry, Fluorescence; Uncoupling Agents; Vanadates

Estrogens are neuroprotective against glutamate excitotoxicity caused by an excessive rise in intracellular calcium ([Ca(2+)](i)). In this study, we demonstrate that 17 beta-estradiol (E(2)) treatment of hippocampal neurons attenuated the excitotoxic glutamate-induced rise in bulk-free [Ca(2+)](i) despite potentiating the influx of Ca(2+) induced by glutamate. E(2)-induced attenuation of bulk-free [Ca(2+)](i) depends on mitochondrial sequestration of Ca(2+), which is blocked in the presence of the combination of rotenone and oligomycin or in the presence of antimycin, which collapse the mitochondrial membrane potential, thereby preventing mitochondrial Ca(2+) transport. Release of mitochondrial Ca(2+) by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) after excitotoxic glutamate treatment resulted in a greater [Ca(2+)](i) in E(2)-treated cells, indicating an E(2)-induced increase in the mitochondrial calcium ([Ca(2+)](m)) load. The increased [Ca(2+)](m) load was accompanied by increased expression of Bcl-2, which can promote mitochondrial Ca(2+) load tolerance. These findings provide a mechanism of E(2)-induced neuronal survival by attenuation of excitotoxic glutamate [Ca(2+)](i) rise via increased mitochondrial sequestration of cytosolic Ca(2+) coupled with an increase in Bcl-2 expression to sustain mitochondrial Ca(2+) load tolerance and function.

Topics: Animals; Antimycin A; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Cytoplasm; Cytosol; Estrogens; Glutamates; Glutamic Acid; Hippocampus; Ionophores; Membrane Potentials; Mitochondria; Neurons; Oligomycins; Phenotype; Proto-Oncogene Proteins c-bcl-2; Rats; Rotenone; Time Factors; Uncoupling Agents

Dependence on mitochondrial membrane potential (deltapsim) of hydrogen peroxide formation of in situ mitochondria in response to inhibition of complex I or III was studied in synaptosomes. Blockage of electron flow through complex I by rotenone or that through complex III by antimycin resulted in an increase in the rate of H2O2 generation as measured with the Amplex red assay. Membrane potential of mitochondria was dissipated by either FCCP (250 nM) or DNP (50 microM) and then the rate of H2O2 production was followed. Neither of the uncouplers had a significant effect on the rate of H2O2 production induced by rotenone or antimycin. Inhibition of the F0F1-ATPase by oligomycin, which also eliminates deltapsim in the presence of rotenone and antimycin, respectively, was also without effect on the ROS formation induced by rotenone and only slightly reduced the antimycin-induced H2O2 production. These results indicate that ROS generation of in situ mitochondria in nerve terminals in response to inhibition of complex I or complex III is independent of deltapsim. In addition, we detected a significant antimycin-induced H2O2 production when the flow of electrons through complex I was inhibited by rotenone, indicating that the respiratory chain of in situ mitochondria in synaptosomes has a substantial electron influx distal from the rotenone site, which could contribute to ROS generation when the complex III is inhibited.

Topics: Animals; Antimycin A; Electron Transport Complex I; Electron Transport Complex III; Guinea Pigs; Hydrogen Peroxide; In Vitro Techniques; Membrane Potentials; Mitochondria; Mitochondrial Proton-Translocating ATPases; Oligomycins; Reactive Oxygen Species; Rotenone; Synaptosomes

Mitochondrial dysfunction can lead to diverse cellular and organismal responses. We used DNA microarrays to characterize the transcriptional responses to different mitochondrial perturbations in Saccharomyces cerevisiae. We examined respiratory-deficient petite cells and respiratory-competent wild-type cells treated with the inhibitors of oxidative phosphorylation antimycin, carbonyl cyanide m-chlorophenylhydrazone, or oligomycin. We show that respiratory deficiency, but not inhibition of mitochondrial ATP synthesis per se, induces a suite of genes associated with both peroxisomal activities and metabolite-restoration (anaplerotic) pathways that would mitigate the loss of a complete tricarboxylic acid cycle. The array data suggested, and direct microscopic observation of cells expressing a derivative of green fluorescent protein with a peroxisomal matrix-targeting signal confirmed, that respiratory deficiency dramatically induces peroxisome biogenesis. Transcript profiling of cells harboring null alleles of RTG1, RTG2, or RTG3, genes known to control signaling from mitochondria to the nucleus, suggests that there are multiple pathways of cross-talk between these organelles in yeast.

Topics: Antimycin A; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Citric Acid Cycle; DNA-Binding Proteins; Enzyme Inhibitors; Fungal Proteins; Gene Expression Regulation, Fungal; Gene Silencing; Genome, Fungal; Intracellular Signaling Peptides and Proteins; Mitochondria; Oligomycins; Oligonucleotide Array Sequence Analysis; Peroxisomes; Phosphorylation; Propionates; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors; Transcription, Genetic

The nuclear gene encoding the Sit4 protein phosphatase was identified in the budding yeast Kluyveromyces lactis. K. lactis cells carrying a disrupted sit4 allele are resistant to oligomycin, antimycin, ketoconazole, and econazole but hypersensitive to paromomycin, sorbic acid, and 4-nitroquinoline-N-oxide (4-NQO). Overexpression of SIT4 leads to an elevation in resistance to paromomycin and to lesser extent tolerance to sorbic acid, but it has no detectable effect on resistance to 4-NQO. These observations suggest that the Sit4 protein phosphatase has a broad role in modulating multidrug resistance in K. lactis. Expression or activity of a membrane transporter specific for paromomycin and the ABC pumps responsible for 4-NQO and sorbic acid would be positively regulated by Sit4p. In contrast, the function of a Pdr5-type transporter responsible for ketoconazole and econazole extrusion, and probably also for efflux of oligomycin and antimycin, is likely to be negatively regulated by the phosphatase. Drug resistance of sit4 mutants was shown to be mediated by ABC transporters as efflux of the anionic fluorescent dye rhodamine 6G, a substrate for the Pdr5-type pump, is markedly increased in sit4 mutants in an energy-dependent and FK506-sensitive manner.

Topics: 4-Nitroquinoline-1-oxide; Antifungal Agents; Antimycin A; Drug Resistance, Multiple; Econazole; Genetic Complementation Test; Genotype; Ketoconazole; Kluyveromyces; Molecular Sequence Data; Oligomycins; Paromomycin; Phosphoprotein Phosphatases; Protein Phosphatase 2; Restriction Mapping; Saccharomyces cerevisiae Proteins; Sorbic Acid; Tacrolimus

The effects of mitochondrial uncoupling on the calcium homeostasis of prostatic cells were investigated using the prostatic cancer cell line LNCaP and indo-1 spectrofluorimetry. Carbonyl cyanide m-chloro-phenylhydrazone (CCCP) was used as uncoupler. Resting LNCaP cells responded to CCCP by a biphasic increase in [Ca2+]i. The first phase of increase which corresponded to the release of a mitochondrial CCCP-sensitive Ca2+ store was followed by a second increase phase consisting of Ca2+ influx through the plasma membrane. The relationship between the CCCP- and the InsP3-sensitive stores was investigated using thapsigargin (TG). The release part of the Ca2+ response to TG was reduced in a time-dependent manner by previous exposure of the cells to CCCP, suggesting that CCCP also acts on non-mitochondrial stores. Our results show that CCCP releases Ca2+ from both mitochondrial and non-mitochondrial stores in prostatic cells. The possible mechanisms of these effects are discussed.

Topics: Adenosine Triphosphate; Antimycin A; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane; Enzyme Inhibitors; Fluorescent Dyes; Humans; Indoles; Male; Mitochondria; Oligomycins; Prostatic Neoplasms; Spectrometry, Fluorescence; Thapsigargin; Time Factors; Tumor Cells, Cultured; Uncoupling Agents

We have previously shown that exposure of Clone 9 cells to hypoxia, cyanide, or azide results in an acute stimulation of glucose transport that is largely mediated by "activation" of glucose transporter (Glut1) sites preexisting in the plasma membrane. However, it is not known whether inhibition of oxidative phosphorylation only at its terminal step, or at any of its steps, leads to the glucose transport response. Hence, the effect of azide (5 mM), rotenone (1 microM), rotenone (1 microM) plus thenoyltrifluoroacetone (TTFA) (5 microM), antimycin A (0.3 microM), dinitrophenol (0.25 mM), carbonyl cyanide m-chlorophenylhydrazone (CCCP) (2.5 microM), and oligomycin B (0.15 microM) on glucose transport was determined. All of the above agents elicited a similar approximately 4-fold stimulation of cytochalasin B (CB)-inhibitable 3-O-methyl glucose (3-OMG) uptake in Clone 9 cells. The stimulatory effect of azide on 3-OMG uptake was not inhibited by antioxidants 2-mercaptopropionyl glycine (1.2 mM) and 1,10-phenanthroline (40 microM), while, in contrast, the antioxidants attenuated the stimulation of glucose transport in response to 250 microM H(2)O(2) by approximately 50%. To differentiate between an increase in the number of functional Glut1 sites in the plasma membrane (in the absence of "translocation") versus an increase in the "intrinsic activity" of Glut1, the effect of azide on the energy of activation (E(a)) of glucose transport was measured. The E(a) was determined by measuring the rate of CB-inhibitable 3-OMG uptake at 24.0, 28.0, 35. 0, and 40 degrees C. The E(a) of control Clone 9 cells and of cells exposed to 10 mM azide for 2 h was 32,530 +/- 1830 and 31,220 +/- 600 J/mol, respectively (P > 0.1), while the rate of CB-inhibitable 3-OMG uptake was 9.3 +/- 0.7-fold higher in azide-treated cells. It is concluded that (i) inhibition of oxidative phosphorylation, at any of its steps, leads to a stimulation of glucose transport, and (ii) the mechanism of stimulation of glucose transport in response to azide appears to be predominately mediated by an apparent increase in the number of functional Glut1 sites in the plasma membrane (instead of an increase in their "intrinsic activity"), suggesting an "unmasking" mechanism.

Topics: Animals; Antimycin A; Azides; Biological Transport; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Chelating Agents; Clone Cells; Dinitrophenols; Glucose; Glucose Transporter Type 1; Liver; Mice; Monosaccharide Transport Proteins; Oligomycins; Oxidative Phosphorylation; Rotenone; Thenoyltrifluoroacetone; Uncoupling Agents

This study used fluorescent indicator dyes to measure changes in cytosolic and mitochondrial [Ca(2+)] produced by physiological stimulation of lizard motor nerve terminals. During repetitive action potential discharge at 10-50 Hz, the increase in average cytosolic [Ca(2+)] reached plateau at levels that increased with increasing stimulus frequency. This stabilization of cytosolic [Ca(2+)] was caused mainly by mitochondrial Ca(2+) uptake, because drugs that depolarize mitochondria greatly increased the stimulation-induced elevation of cytosolic [Ca(2+)], whereas blockers of other Ca(2+) clearance routes had little effect. Surprisingly, during this sustained Ca(2+) uptake the free [Ca(2+)] in the mitochondrial matrix never exceeded a plateau level of approximately 1 microM, regardless of stimulation frequency or pattern. When stimulation ceased, matrix [Ca(2+)] decreased over a slow ( approximately 10 min) time course consisting of an initial plateau followed by a return to baseline. These measurements demonstrate that sustained mitochondrial Ca(2+) uptake is not invariably accompanied by progressive elevation of matrix free [Ca(2+)]. Both the plateau of matrix free [Ca(2+)] during stimulation and its complex decay after stimulation could be accounted for by a model incorporating reversible formation of an insoluble Ca salt. This mechanism allows mitochondria to sequester large amounts of Ca(2+) while maintaining matrix free [Ca(2+)] at levels sufficient to activate Ca(2+)-dependent mitochondrial dehydrogenases, but below levels that activate the permeability transition pore.

Topics: Action Potentials; Animals; Antimycin A; Axons; Caffeine; Calcium; Carbachol; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Clonazepam; Cytosol; Electric Stimulation; In Vitro Techniques; Indoles; Intracellular Membranes; Lizards; Membrane Potentials; Mitochondria, Muscle; Models, Biological; Motor Endplate; Motor Neurons; Muscle Contraction; Muscle, Skeletal; Nerve Endings; Neuromuscular Junction; Oligomycins; Thiazepines; Tubocurarine

Agonist-evoked cytosolic Ca(2+) spikes in mouse pancreatic acinar cells are specifically initiated in the apical secretory pole and are mostly confined to this region. The role played by mitochondria in this process has been investigated. Using the mitochondria-specific fluorescent dyes MitoTracker Green and Rhodamine 123, these organelles appeared as a bright belt concentrated mainly around the secretory granule area. We tested the effects of two different types of mitochondrial inhibitor on the cytosolic Ca(2+) concentration using simultaneous imaging of Ca(2+)-sensitive fluorescence (Fura 2) and electrophysiology. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied in the presence of the Ca(2+)-releasing messenger inositol 1,4, 5-trisphosphate (IP(3)), the local repetitive Ca(2+) responses in the granule area were transformed into a global rise in the cellular Ca(2+) concentration. In the absence of IP(3), CCCP had no effect on the cytosolic Ca(2+) levels. Antimycin and antimycin + oligomycin had the same effect as CCCP. Active mitochondria, strategically placed around the secretory pole, block Ca(2+) diffusion from the primary Ca(2+) release sites in the granule-rich area in the apical pole to the basal part of the cell containing the nucleus. When mitochondrial function is inhibited, this barrier disappears and the Ca(2+) signals spread all over the cytosol.

Topics: Animals; Antimycin A; Calcium Signaling; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cytoplasmic Granules; Hydrogen-Ion Concentration; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Mice; Mitochondria; Models, Biological; Oligomycins; Pancreas; Uncoupling Agents

This article describes the first detailed analysis of mitochondrial electron transfer and oxidative phosphorylation in the pathogenic filamentous fungus, Gaeumannomyces graminis var. tritici. While oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly, maintenance of a Deltapsi across the mitochondrial inner membrane was unaffected by cyanide but sensitive to antimycin A and SHAM when succinate was added as the respiratory substrate. As a result, ATP synthesis through complex V was demonstrated to be sensitive to these two inhibitors but not to cyanide. Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of both the monomeric and dimeric forms of this oxidase. Overall this study demonstrates that a novel respiratory chain utilizing the terminal oxidases, cytochrome c oxidase and alternative oxidase, are present and constitutively active in electron transfer in G. graminis tritici. These results are discussed in relation to current understanding of fungal electron transfer and to the possible contribution of alternative redox centers in ATP synthesis.

Topics: Antifungal Agents; Antimycin A; Ascomycota; Carboxin; Cytochromes; Electron Transport; Microbial Sensitivity Tests; Oligomycins; Oxygen Consumption; Potassium Cyanide; Salicylamides; Triticum

The role of ATP in both the activation of store-operated Ca2+ current ICRAC and in Ca2+-dependent vesicular fusion was examined in a study of rat basophilic leukaemia (RBL) cells using the whole-cell patch-clamp technique. Fusion was monitored via changes in plasma membrane capacitance. Following a decrease in the levels of intracellular ATP, achieved using the mitochondrial poison antimycin and the ATP synthase inhibitor oligomycin, as well as a reduction of glycolysis by removal of external glucose, ICRAC activated in a manner similar to control cells when stores are depleted by dialysis with a pipette solution containing either inositol 1,4, 5-trisphosphate (InsP3) or ionomycin together with a high concentration of EGTA. Dialysis of cells for 150 s with the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) (2 mM) in addition to the mitochondrial inhibitors also failed to prevent activation of ICRAC following external application of ionomycin and thapsigargin, when compared with control recordings obtained with 2 mM ATP instead. Ca2+-dependent vesicular fusion was triggered by dialysing cells with 10 microM Ca2+ and guanosine-5'-O-(3-thiotriphosphate (GTP[gamma-S]). The capacitance increase was unaffected by inhibition of glycolysis, mitochondrial inhibitors or dialysis with either AMP-PNP or adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) instead of ATP. We conclude that ATP hydrolysis does not seem to be necessary for the activation of ICRAC or for the capacitance increases elicited by high concentrations of intracellular Ca2+.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Antimycin A; Calcium; Cell Membrane; Electric Conductivity; Enzyme Inhibitors; Leukemia, Basophilic, Acute; Membrane Fusion; Oligomycins; Patch-Clamp Techniques; Proton-Translocating ATPases; Rats; Tumor Cells, Cultured

Compared with parental GC3/c1 human colon adenocarcinoma cells, which are diarylsulfonylurea (DSU)-sensitive cells, the DSU-resistant clone LYC5 demonstrates 4.2-, 12.8-, and 5.3- fold increase in sensitivity to the mitochondrial toxins rotenone, antimycin, and oligomycin, respectively. Studies with hybrids formed by fusion of parental GC3/c1 cells with LYC5 cells have indicated that resistance to antitumor DSUs and collateral sensitivity to mitochondrial toxins are recessive and therefore potentially linked. To examine this, we transfected a cDNA library from GC3/c1 cells, constructed in pcDNA3, into LYC5 cells. G418-resistant colonies were selected and further selected in a single step for resistance to rotenone (100 nm). Individual colonies (designated T5LR) were expanded and tested for sensitivity to mitochondrial toxins, antitumor DSU agents (LY195779 and LY186391) that demonstrate a 45-50-fold differential potency against GC3/c1, LYC5 cells, and the antimitotic agent vincristine. Results demonstrate that resistance to mitochondrial toxins rotenone, antimycin, and oligomycin can be transferred without conferring a DSU-sensitive phenotype. Furthermore, in T5LR clones, resistance to mitochondrial toxins was not associated with increased resistance to vincristine or increased P-glycoprotein expression, supporting the contention that resistance to these agents is independent of P-glycoprotein. Southern blot analysis of T5LR clones demonstrated unique integration sites for the neomycin phosphotransferase gene into genomic DNA in clones 4 and 9, indicating independent derivation. Analysis of clones 4, 6, and 9 with use of polymerase chain reaction demonstrated a cDNA insert of approximately 1.0 kilobase.

Topics: Antimycin A; Antineoplastic Agents; Base Sequence; Drug Resistance; Humans; Mitochondria; Molecular Sequence Data; Oligomycins; Rotenone; Sulfonylurea Compounds; Tumor Cells, Cultured; Vincristine

The purpose of our work was to study the relationship between glutamate (GLU)-induced mitochondrial depolarization and deterioration of neuronal Ca2+ homeostasis following a prolonged GLU challenge. The experiments were performed on cultured rat cerebellar granule cells using the fluorescent probes, rhodamine 123 and fura-2. All the cells, in which 100 microM GLU (10 microM glycine, 0 Mg2+) induced only relatively slight mitochondrial depolarization (1.1-1.3-fold increase in rhodamine 123 fluorescence), retained their ability to recover [Ca2+]i following a prolonged GLU challenge. In contrast, the cells in which GLU treatment induced pronounced mitochondrial depolarization (2-4-fold increase in rhodamine 123 fluorescence), exhibited a high Ca2+ plateau in the post-glutamate period. Application of 3-5 mM NaCN or 0.25-1 microM FCCP during this Ca2+ plateau phase usually failed to produce a further noticeable increase in [Ca2+]i. Regression analysis revealed a good correlation (r2 = 0.88 +/- 0.03, n = 19) between the increase in the percentage of rhodamine 123 fluorescence and the post-glutamate [Ca2+]i. Collectively, the results obtained led us to conclude that the GLU-induced neuronal Ca2+ overload was due to the collapse of the mitochondrial potential and subsequent ATP depletion.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Cerebellum; Fura-2; Glutamic Acid; Intracellular Membranes; Mitochondria; Neurons; Oligomycins; Rats; Rats, Wistar; Rhodamine 123; Rhodamines; Sodium Cyanide

Treatment of isolated mitochondria with Ca2+ and inorganic phosphate (Pi) induces an inner membrane permeability that appears to be mediated through a cyclosporin A (CsA)-inhibitable Ca(2+)-dependent pore. Isolated mitochondria during inner membrane permeability undergo rapid efflux of matrix solutes such as glutathione as GSH and Ca2+, loss of coupled functions, and large amplitude swelling. Permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability, has been observed. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing Ca2+ and Pi abolished large amplitude swelling of mitochondria. The GSH status during a Ca(2+)- and Pi-dependent mechanism of mitochondrial GSH release in isolated mitochondria was influenced significantly by metabolic inhibitors of the respiratory chain but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial GSH and Ca2+. The release of GSH was inhibited by the addition of CsA, a potent inhibitor of permeability transition. Under these conditions we did not find GSSG; however, rapid oxidation of pyridine nucleotides and depletion of ATP and ADP with conversion to AMP occurred. The addition of CsA, prevented the oxidation of pyridine nucleotides and depletion of ATP and ADP. Since NADH and NADPH were extensively oxidized, protection against oxidative stress is reflected in maintenance of GSH and not observable lipid peroxidation. Evidence from transmission electron microscopy analysis, combined with the GSH release data, indicate that permeability transition can be observed in the absence of large amplitude swelling.

Topics: Adenine Nucleotides; Animals; Antimycin A; Calcium; Cyclosporine; Glutathione; Intracellular Membranes; Male; Microscopy, Electron; Mitochondria, Liver; NAD; NADP; Oligomycins; Permeability; Phosphates; Rats; Rats, Sprague-Dawley; Sulfides

We have investigated the effects of imposing an ATP demand, generated by the addition of lactate, on hepatocytes isolated from fasted normal and streptozocin-induced diabetic rats. The stimulation of O2 consumption upon lactate addition was much greater in hepatocytes from diabetic rats, as a result of a lactate-induced stimulation of beta-oxidation that was not observed in control cells. This lactate-induced increment in beta-oxidation was extremely sensitive to inhibition by low levels of a number of inhibitors of energy transduction, implying that the increment was tightly coupled to ATP synthesis. Such sensitivity of the beta-oxidative pathway to the addition of similar low concentrations of these inhibitors was not seen in control cells. Inhibitors of the gluconeogenic pathway were also more effective in decreasing beta-oxidation in cells from diabetic animals than in cells from normal rats. The increment in beta-oxidation was not accompanied by increased rates of glucose synthesis, fatty acid esterification or ureogenesis. We propose that it may be associated with higher rates of glucose cycling in cells from diabetic rats.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Diabetes Mellitus, Experimental; Fasting; Fatty Acids; Gluconeogenesis; Lactates; Lactic Acid; Liver; Male; Mitochondrial Trifunctional Protein; Multienzyme Complexes; Oligomycins; Oxygen Consumption; Rats; Rats, Wistar

Diarylsulfonylurea (DSU) antitumor agents represent a new class of oncolytic compounds with an unknown, potentially novel, mechanism of action. At high concentrations of several of these agents, cytotoxicity appears to be a consequence of uncoupling of mitochondria. However, the mechanism of action at pharmacologically achievable concentrations is unknown. To further study these agents a subline of human colon carcinoma, GC3/c1, was selected for resistance to N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (ISCU) (Sulofenur). This clone (designated LYC5) was stably resistant for 2 years in the absence of selection pressure and was characterized for cross-resistance to other antitumor DSU and therapeutically used oncolytic agents. LYC5 was cross-resistant to six of seven DSU analogues examined when cells were exposed to drugs for 7 days. However, the degree of resistance was inversely related to the potency of the individual DSU against the parental GC3/c1 clone. Consequently, against LYC5 cells there was a relatively narrow range for concentrations inhibiting colony formation by 50% (4-fold), compared with that in GC3/c1 cells (12-fold range). With a single exception, each DSU examined caused uncoupling of oxidative phosphorylation in isolated mitochondria at 50 microM, and data suggest that cytotoxicity in LYC5 cells may be a consequence of mitochondrial impairment. In contrast, LYC5 cells were collaterally sensitive to the mitochondrial toxins rotenone, antimycin, and oligomycin, by 11.4-, 7.2-, and 36.9-fold respectively. LYC5 cells were also collaterally sensitive to vincristine (7.7-fold), Actinomycin D (5.9-fold), and rhodamine-123 (10.5-fold), agents associated with P-glycoprotein (Pgp)-mediated multidrug resistance (MDR). LYC5 cells were slightly more sensitive to Melphalan and doxorubicin (2.8- and 2.3-fold, respectively) but not to cisplatin or dideazatetrahydrofolic acid. Collateral sensitivity to vincristine and Actinomycin D was consistent with decreased Pgp levels in LYC5 cells. Immunohistochemical staining and Western blotting with anti-Pgp antibodies indicated an 8-fold reduction in Pgp levels in LYC5 cells, relative to expression in parental GC3/c1 cells. Consequently, association of mitochondrial toxins with resistance in MDR KB8-5 cells was examined in the presence or absence of the MDR-reversing agent verapamil. KB8-5 cells had equal or greater sensitivity, compared with parental KB3-1 cells, to rotenone, antimycin, and oligomycin and al

Topics: Antimycin A; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carrier Proteins; Drug Interactions; Drug Resistance; Humans; Membrane Glycoproteins; Mitochondria; Oligomycins; Rotenone; Sulfonylurea Compounds; Tumor Cells, Cultured; Verapamil

Treatment of isolated mitochondria with calcium and inorganic phosphate induces inner membrane permeability that is thought to be mediated through a non-selective, calcium-dependent pore. The inner membrane permeability results in the rapid efflux of small matrix solutes such as glutathione and calcium, loss of coupled functions, and large amplitude swelling. We have identified conditions of permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing calcium and inorganic phosphate abolished large-amplitude swelling of mitochondria but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial glutathione and calcium. The release of both glutathione and calcium was inhibited by the addition of cyclosporin A, a potent inhibitor of permeability transition. Transmission electron microscopy analysis, combined with the glutathione and calcium release data, indicate that permeability transition can be observed in the absence of large-amplitude swelling. Permeability transition occurring both with and without large-amplitude swelling was accompanied by a collapse of the membrane potential. We conclude that cyclosporin A-sensitive permeability transition can occur without obvious morphological changes such as large-amplitude swelling. Monitoring the cyclosporin A-sensitive release of concentrated endogenous matrix solutes, such as GSH, may be a sensitive and useful indicator of permeability transition.

Topics: Animals; Antimycin A; Biological Transport; Calcium; Cyclosporine; Glutathione; Male; Microscopy, Electron; Mitochondria, Liver; Mitochondrial Swelling; Oligomycins; Onium Compounds; Permeability; Phosphates; Rats; Rats, Sprague-Dawley; Sulfides; Time Factors; Trityl Compounds

Intracellular magnesium is intimately associated with adenosine triphosphate (ATP) concentrations and energy utilization. We determined adenine nucleotide concentrations (ATP, adenosine diphosphate, and adenosine triphosphate) and the associated changes in intracellular free Mg2+ ([Mg2+]i) by high-performance liquid chromatography and fluorescent methods, respectively. Various mitochondrial inhibitors were used to deplete intracellular ATP and alter energy charge in epithelial cells. The opossum kidney (OK) cell line was used as a prototypic renal epithelial cell. These agents markedly deplete intracellular ATP levels with modest changes in [Mg2+]i and [Ca2+]i. Because these agents have disparate actions, it is likely that these changes were due to alterations in ATP rather than to selective drug effects. Cyanide resulted in a rapid (within 2 minutes) fall in ATP from 25.85 to 10.58 nmol/mg protein or about 3 mmol/L, whereas [Mg2+]i increased gradually (10 minutes), from 513 +/- 7 to 1096 +/- 105 mumol/L [Ca2+]i increased from 109 +/- 12 to 153 +/- 10 nmol/L within 20 seconds, then returned to basal concentrations. The changes in ATP, Mg2+, and Ca2+ were not altered by removing external Na+o, adding ruthenium red, or treating with vanadate. Antimycin diminished ATP levels in a manner similar to the effect of cyanide, but by contrast [Mg2+]i decreased to 436 +/- 13 mumol/L and [Ca2+]i transiently increased. These studies indicate that we are able to distinguish Mg2+ movements from those of Ca2+ by fluorescent techniques and suggest that intracellular regulation of [Mg2+]i is distinctive from that of [Ca2+]i. Oligomycin resulted in marked and rapid falls in [ATP]i with disproportionate increases in [Mg2+]i. The response of magnesium-depleted cells (basal [Mg2+]i, 231 +/- 10 mumol/L) after inhibitor-induced energy depletion was similar to that of control cells. These studies suggest that large changes in intracellular ATP levels do not markedly alter intracellular [Mg2+]i control and, in turn, that intracellular free Mg2+ is not a limiting factor in ATP metabolism after energy depletion with chemical hypoxia.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Cell Hypoxia; Cyanides; Cytosol; Intracellular Membranes; Kidney; Magnesium; Oligomycins; Osmolar Concentration

We have used RNase protection to measure oxygen-dependent changes in erythropoietin (EPO) mRNA in isolated perfused kidneys and to compare the effect of hypoxia with the response to inhibitors of oxidative phosphorylation. In well-oxygenated kidneys perfused for 2 h at 12 ml/min, with hematocrit of 0.09 +/- 0.005 and PO2 of 443 +/- 67 mmHg, EPO mRNA levels were similar to the baseline levels measured in nonperfused contralateral kidneys from the same animals. When perfusions were performed under identical conditions but at a PO2 of 32 +/- 4 mmHg, EPO mRNA increased approximately 16-fold. In contrast, graded concentrations of cyanide (10, 100, and 300 microM and 1 mM), antimycin (0.01, 0.1, 0.5, and 1 microM), and oligomycin (0.01, 0.1, and 1 microM) did not alter EPO mRNA in well-oxygenated perfused kidneys. However, in kidneys perfused at low PO2 with a high concentration of each inhibitor, EPO mRNA levels were increased, demonstrating that the ability to respond to hypoxia was retained. Thus inhibitors of oxidative phosphorylation did not mimic the effects of hypoxia, indicating that oxygen-dependent expression of the EPO gene in the kidney is not effected through hypoxic compromise of oxidative phosphorylation.

Topics: Animals; Antimycin A; Erythropoietin; Gene Expression; Kidney; Male; Oligomycins; Oxygen; Potassium Cyanide; Rats; Rats, Inbred Strains; RNA, Messenger; Uncoupling Agents

The respiratory capacities of hepatocytes, derived from hypothyroid, euthyroid and hyperthyroid rats, have been compared by measuring rates of oxygen uptake and by titrating components of the respiratory chain with specific inhibitors. Thyroid hormone increased the maximal rate of substrate-stimulated respiration and also increased the degree of ionophore-stimulated oxygen uptake. In titration experiments, similar concentrations of oligomycin or antimycin were required for maximal inhibition of respiration regardless of thyroid state, suggesting that the changes in respiratory capacity were not the result of variation in the amounts of ATP synthase or cytochrome b. However, less rotenone was required for maximal inhibition of respiration in the hypothyroid state than in cells from euthyroid or hyperthyroid rats, implying that hepatocytes from hypothyroid animals contain less NADH dehydrogenase. The concentration of carboxyatractyloside necessary for maximal inhibition of respiration was 100 microM in hepatocytes from hypothyroid rats, but 200 microM and 300 microM in hepatocytes from euthyroid and hyperthyroid rats, respectively, indicating a possible correlation between levels of thyroid hormone and the amount or activity of adenine nucleotide translocase. The increased capacity for coupled respiration in response to thyroid hormone is not associated with an increase in the components of the electron transport chain or ATP synthase, but correlates with an increased activity of adenine nucleotide translocase.

Topics: Animals; Antimycin A; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Hyperthyroidism; Hypothyroidism; Kinetics; Liver; Male; Oligomycins; Oxygen Consumption; Rats; Rats, Inbred Strains; Reference Values; Rotenone; Thyroid Gland; Triiodothyronine

The proton ionophore FCCP was evaluated for use as a selective blocker of mitochondrial Ca2+ sequestration in identified Helisoma neurons in vitro. By use of the Ca2+ indicator fura-2, it was found that application of FCCP evoked a gradual increase in cell body [Ca2+]i that reached a level approximately 3-fold higher than baseline after 60 min. Moreover, FCCP released Ca2+ even when added after mitochondrial stores of Ca2+ had previously been emptied by an alternate method. From these and other experiments, it is concluded that FCCP, in addition to its recognized effect on mitochondrial Ca2+ sequestration, also releases Ca2+ from a non-mitochondrial store and is, therefore, unsuitable for use in an intact neuron to selectively inactivate mitochondrial Ca2+ uptake.

Topics: Animals; Antimycin A; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Mitochondria; Neurons; Oligomycins; Osmolar Concentration; Rotenone; Snails

It is well established that calcium ionophore A 23187 induces acrosome reaction (AcR) of uncapacitated spermatozoa in the presence of extracellular Ca2+ ions. In the present study, we have investigated how extracellular energy substrates (glucose, pyruvate, and lactate) affect the ionophore-induced AcR of guinea pig spermatozoa. It was found that 0.3 microM concentration of A 23187 had the maximum effect to initiate AcR of guinea pig spermatozoa. Virtually no spermatozoa underwent their AcR when incubated in substrate-free modified Tyrode's medium containing 0.3 microM A 23187 and 2 mM Ca2+. At least one exogenous substrate is essential for the ionophore-induced AcR of spermatozoa. As for efficacy of the substrates, lactate was more effective than pyruvate and glucose. However, a better result was observed when lactate was added along with pyruvate. Malonate inhibited the ionophore-induced AcR but not the hyperactivated motility of spermatozoa. The mitochondrial electron transport chain blockers rotenone, antimycin, and oligomycin failed to inhibit AcR, although in the presence of these blockers spermatozoa were unable to show hyperactivated motility. These results suggest that the mitochondrial citric acid cycle, not the electron transport chain, is probably the energy source for ionophore-induced AcR of guinea pig spermatozoa.

Topics: Acrosome; Animals; Antimycin A; Calcimycin; Energy Metabolism; Glucose; Guinea Pigs; Lactates; Male; Malonates; Mitochondria; Oligomycins; Pyruvates; Rotenone

The present studies were performed using primary monolayer cultures of bovine glomerulosa cells to determine whether the elevation in cytosolic calcium concentration produced by angiotensin II was accompanied by an elevation in mitochondrial calcium. Exchangeable mitochondria calcium content was assessed indirectly by measuring the changes in cytosolic calcium concentration and calcium efflux produced by the mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Total mitochondrial calcium content was also assessed directly by atomic absorption spectroscopy. CCCP had a direct effect to promote calcium release from an oligomycin/antimycin-sensitive (mitochondrial) calcium pool in permeabilized cells. In intact cells, CCCP caused rapid reductions in cellular ATP content and the ratio of ATP to ADP. Still, its effects on calcium dynamics were exerted primarily at the mitochondrial level as evidenced by inhibition with ruthenium red, but not dantrolene. As expected, angiotensin II produced a rapid increase in calcium efflux and an equally rapid and sustained increase in cytosolic calcium concentration. Nonetheless, CCCP-stimulated elevations in cytosolic calcium concentration and calcium efflux were reduced by angiotensin II in a concentration-dependent manner. Total mitochondrial calcium content was also lower in angiotensin-treated than in control cells. These results indicate that angiotensin II causes a net decrease in mitochondrial calcium stores. On the basis of these data, it is proposed that alterations in calcium metabolism initiated by angiotensin II are exerted not only at the membrane and cytosolic levels but also at the level of the mitochondria. Changes in mitochondrial calcium dynamics may directly contribute to the regulation of mitochondrial steroidogenic enzymes by angiotensin II.

Topics: Adenosine Triphosphate; Angiotensin II; Animals; Antimycin A; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cattle; Cell Membrane Permeability; Cells, Cultured; Dantrolene; Kinetics; Mitochondria; Oligomycins; Ruthenium Red; Spectrophotometry, Atomic; Zona Glomerulosa

Protein synthesis was investigated in isolated mitochondria under conditions which either inhibited electron transport or uncoupled oxidative phosphorylation. In a medium containing an exogenous source of ATP and oligomycin, an inhibitor of the ATP synthase complex, incorporation of [35S]methionine into proteins is stimulated in the presence of inhibitors of the electron transport chain; substituting uncouplers of oxidative phosphorylation for the latter leads, in contrast, to a decrease in the rate of incorporation of the labeled amino acid into mitochondrial translation products. Studies on the metabolic stability of mitochondrial translation products revealed that "mature" polypeptides made in isolated mitochondria are stable as indicated by the absence of degradation during a 50 min "chase" period. Under conditions which reduce or dissipate the membrane potential, 50-60% of the newly made polypeptides (pulse) are degraded within 50 min. The kinetics of the degradation process for individual mitochondrial gene products reveal that the largest proportion of polypeptides degraded to an acid-soluble form during the chase period are abnormal proteins, likely the result of premature chain termination. Emerging as a common denominator in these studies is a role for a transmembrane potential across the inner membrane in the production of mature "stable" mitochondrial gene products.

Topics: Animals; Antimycin A; Atractyloside; Intracellular Membranes; Kinetics; Membrane Potentials; Methionine; Mitochondria, Heart; Oligomycins; Potassium Cyanide; Protein Biosynthesis; Proteins; Rats; Valinomycin

We have determined whether glycine or glutathione can protect rabbit proximal tubules damaged by chemical inhibitors of oxidative phosphorylation: antimycin A, rotenone, cyanide, oligomycin, or carbonyl cyanide m-chlorophenylhdrazone (CCCP). All the agents severely depleted cell ATP levels within 15 min and caused lethal cell injury, as quantified by lactate dehydrogenase (LDH) release. Glycine and glutathione largely prevented this injury without altering the primary effects of the inhibitors on tubule respiration or the depletion of ATP. Buthionine sulfoximine and 1,3-bis(2-chloroethyl)-1-nitrosourea decreased cell glutathione but did not prevent the protective effects of either glycine or glutathione in tubules treated with rotenone. Protection was sustained during both a 15-min exposure and a 45-min postwash period irrespective of whether the wash removed the agent or mitochondrial function recovered. Cysteine uniquely induced a dramatic recovery of mitochondrial function in tubules washed after treatment with CCCP. These data 1) demonstrate that the cytoprotective effects of glycine previously seen during hypoxia extend to other tubule lesions characterized by severe ATP depletion, 2) emphasize the actions of glycine to preserve cell structural integrity in spite of sustained severe impairment of ATP-generating processes in proximal tubules, and 3) indicate that it is glycine rather than intracellular or extracellular glutathione which mediates protection.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cyanides; Cysteine; Glutathione; Glycine; Kidney Cortex; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Male; Microscopy, Electron; Mitochondria; Nitriles; Oligomycins; Oxidative Phosphorylation; Rabbits; Rotenone

Addition of fatty acids to isolated hepatocytes raised respiration rate by 92% and raised mitochondrial membrane potential (delta psi m) in situ from 155 to 162 mV suggesting that the increased fuel supply had a greater effect on respiration rate than any increases in processes that consumed mitochondrial protonmotive force (delta p). The relationship between delta psi m and respiration rate was changed by addition of fatty acids or lactate, showing that there was also stimulation of delta p-consuming reactions. In the presence of oligomycin the relationship between delta psi m and respiration rate was unaffected by substrate addition, showing that the kinetics of delta p consumption by the H+ leak across the mitochondrial inner membrane were unchanged. The stimulation of delta p consumers by fatty acids therefore must be in the pathways of ATP synthesis and turnover. Inhibition of several candidate ATP-consuming reactions had little effect on basal or fatty acid-stimulated respiration, and the nature of the ATP turnover reactions in hepatocytes remains speculative. We conclude that fatty acids (and other substrates) stimulate respiration in hepatocytes in two distinct ways. They provide substrate for the electron transport chain, raising delta p and increasing the non-ohmic proton leak across the mitochondrial inner membrane and the rate of oxygen consumption. They also directly stimulate an unidentified delta p-consuming reaction in the cytoplasm. They do not work by uncoupling or by stimulation of intramitochondrial ATP-turnover reactions.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Caprylates; Cell Membrane; Cells, Cultured; Female; Glucose; Kinetics; Liver; Membrane Potentials; Mitochondria, Liver; Oligomycins; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Phosphoenolpyruvate Carboxykinase (GTP); Picolinic Acids; Rats; Rats, Inbred Strains

Frozen-stored bovine sperm-pellets of proven fertility were used, and the response to respiratory chain effectors was studied, thus demonstrating the energy conservation capacity. It was further observed that the assayed suspensions used lactate oxidatively, which proves the LDH-X mitochondrial activity (the presence of oxidative substrates is fundamental in capacitation and acrosome reaction processes). The suspensions were treated with 10mM phosphate buffer hypotonic medium to eliminate plasmalema and cytoplasmic content. Lactate respiration was sensitive to respiratory chain effectors, such as oligomycin and antimycin. To evaluate the LDH-X contribution to mitochondrial respiration, lipoate dehydrogenase was inhibited through 5-methoxyindole-2-carboxylic acid (MICA) in the presence of pyruvate-malate and citrate-malate, obtaining with the addition of lactate, oxygen uptakes of 18% and 51% with respect to respiration with the mentioned substrates. In the MICA dose-effect curve, a major sensitivity to inhibitor in active state mitochondrial respiration is obtained when pyruvate-malate is used. Lactate competence with pyruvate by mitochondrial LDH-X was observed. The results obtained would allow the thorough study of the necessity of oxidative energy in the capacitation and fertilization processes, and of the LDH-X role in frozen-stored bovine sperm.

Topics: Animals; Antimycin A; Cattle; Cryopreservation; Dihydrolipoamide Dehydrogenase; Energy Metabolism; Indoles; L-Lactate Dehydrogenase; Lactates; Lactic Acid; Male; Mitochondria; Oligomycins; Oxidation-Reduction; Oxygen Consumption; Phosphorylation; Spermatozoa; Tissue Preservation

The mechanism of T3 entry into cells was studied in undifferentiated NB41A3 neuroblasts and after differentiation with 0.5 mM sodium butyrate. In undifferentiated neuroblasts, cell uptake of labeled L-T3 at 2 h was reduced to 43% of the control value by excess L-T3, whereas only 5.9% of D-T3 uptake was saturable. After incubation of intact cells with labeled hormone, the nuclei contained 3.8% of the total cellular L-T3 and 4.3% of D-T3. Thus, L-T3 nuclear uptake was 3 times higher than D-T3. Kinetic analysis of the initial rate of uptake of L-T3 by the cells gave a Km of 1.25 nM and a maximum velocity of 2.38 fmol/min.10(6) cells. The initial rate of D-T3 uptake was not saturable. Inhibitors of ATP production (antimycin and oligomycin) as well as monodansylcadaverine virtually abolished saturable cell uptake and decreased nuclear uptake more than total cell uptake, suggesting that the saturable component of uptake into the cells is the major source of nuclear L-T3. After differentiation by butyrate, both cell uptake and nuclear uptake of L-T3 increased. The apparent affinity (Ka) of the nuclear T3 receptors was determined in intact cells and compared with the Ka measured with isolated nuclei. In undifferentiated cells, the apparent Ka was 3-fold higher than the true Ka, presumably due to a step-up in the free T3 concentration in the cytosol compared to that in the incubation medium. After butyrate exposure, the apparent nuclear Ka was decreased to less than 1.5 times the control value, but Ka in isolated nuclei was unchanged. The number of nuclear receptors, however, was increased by butyrate in both intact cell and isolated nuclei experiments. These results indicate that butyrate exerts separate effects on the number of nuclear receptors and saturable T3 transport in mouse neuroblasts.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Biological Transport; Butyrates; Butyric Acid; Cadaverine; Cell Differentiation; Cell Nucleus; Cytosol; Glucose; Kinetics; Mice; Neuroblastoma; Oligomycins; Stereoisomerism; Triiodothyronine; Tumor Cells, Cultured

In isolated mitochondria from HeLa cells, the ATP requirements for mitochondral DNA (mtDNA) transcription and RNA processing can be satisfied by either endogenous synthesis, mainly through oxidative-phosphorylation, or by exogenous supply. The pattern of RNA synthesis changes dramatically depending upon the level of ATP available. At the low intramitochondrial ATP levels produced from endogenous ADP in the presence of an oxidizable substrate and phosphate, the mRNA species are labeled to a substantial extent, whereas there is only a marginal labeling of the rRNA species and light (L) strand transcripts. By contrast, high ATP levels, either provided exogenously or produced endogenously in the presence of an oxidizable substrate, phosphate, and exogenous ADP, strongly stimulate rRNA synthesis (about 10-fold) and light (L) strand transcription (greater than 10-fold), with only a slight increase in mRNA synthesis.

Topics: Adenosine Triphosphate; Antimycin A; DNA, Mitochondrial; Endonucleases; HeLa Cells; Humans; Mitochondria; Oligomycins; Phosphates; Pyruvates; Pyruvic Acid; RNA; RNA, Messenger; RNA, Mitochondrial; RNA, Ribosomal; Rotenone; Single-Strand Specific DNA and RNA Endonucleases; Transcription, Genetic

Rabbit aortic strips were used to investigate the effect of mitochondrial inhibitors on basal (unstimulated) endothelium dependent relaxation. Since haemoglobin inhibits and the cyclic GMP phosphodiesterase inhibitor, MB22948, amplifies endothelium dependent relaxation they were used to provide evidence of basal activity of endothelium derived relaxing factor (EDRF). Basal activity was not inhibited by incubation with any of three differently acting inhibitors of mitochondrial ATP generation. The mechanism underlying basal EDRF production may thus differ from that of stimulated EDRF production, which is abolished by these mitochondrial inhibitors.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Antimycin A; Aorta; Biological Products; Deoxyglucose; Hemoglobins; In Vitro Techniques; Mitochondria, Muscle; Nitric Oxide; Oligomycins; Purinones; Rabbits; Rotenone; Serotonin; Vasoconstriction

Metabolic and permeability properties of enterocytes isolated by treatment of rat small intestine with hyaluronidase or EDTA were compared. No significant difference was observed in the ability of the two types of cell to produce lactate from glucose. However, while cells obtained with hyaluronidase accumulate alpha-methylglucoside, cells obtained with EDTA were unable to accumulate the sugar above the medium concentrations. When resuspended in a medium designed to resemble the intracellular medium, potentiometric measurements showed that cells obtained with hyaluronidase released Ca2+ to the medium while cells obtained with EDTA accumulated it. Using 45Ca transport assays, this was shown to be an ATP-dependent process, the accumulated 45Ca being totally released by the addition of the ionophore A23187. When cells obtained with EDTA were resuspended in a medium containing concentrations of free Ca2+ higher that 10 microM, the uptake was partially inhibited by sodium orthovanadate and also by oligomycin and antimycin. At free Ca2+ concentrations lower than 1 microM, the accumulation was inhibited up to 87% by sodium orthovanadate while mitochondrial inhibitors inhibited only 5%. Thus, it appears that during their preparation cells obtained with hyaluronidase retain their integrity while cells obtained with EDTA become permeable to Ca2+ and other ions. The usefulness of both types of preparation in metabolic and transport studies is discussed.

Topics: Animals; Antimycin A; Calcimycin; Calcium; Calcium-Transporting ATPases; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane Permeability; Edetic Acid; Glycolysis; Hyaluronoglucosaminidase; Intestinal Absorption; Intestine, Small; Methylglucosides; Nigericin; Oligomycins; Rats; Vanadates; Vanadium

After inducing haemodynamic cardiac overload in rabbits, the authors studied in several stages (1-14 months) the calcium transport activity of the mitochondrial and sarcoplasmic myocardial fractions using labelled 45CaCl2. A coincidence was found between changes in myocardial contractility and changes in calcium transport activity of intracellular organelles. A possible important role of mitochondria in this adaptive process was also documented. Since the calcium transport capacity of the sarcoplasmic reticulum progressively decreases (with the exception of the earliest stages following overload induction), it seems that increased myocardial contractility ensures enhanced Ca transport activity of the mitochondria. Myocardial contractility drops only at the time when the Ca transport activity of the mitochondria decreases. Since these changes occur already at the time of regression of myocardial hypertrophy, which precedes heart failure, it can be assumed that they are causally connected with the reduced contractility of a failing heart.

Topics: Animals; Antimycin A; Calcium; Calcium-Transporting ATPases; Cardiomegaly; Chemical Fractionation; Heart Failure; Intracellular Membranes; Mitochondria, Heart; Myocardium; Oligomycins; Rabbits; Rotenone; Sarcoplasmic Reticulum

In brain, heart and kidney, cell work in the absence of oxygen has been thought to precipitate anoxic damage by increasing the rate of depletion of cellular energy stores. In the medullary thick ascending limb of isolated perfused rat kidneys, however, reduction of ATP synthesis by a variety of mitochondrial or metabolic inhibitors caused ATP depletion comparable to that produced by oxygen deprivation but did not reproduce the lesions of anoxia. In these cells, unrestrained mitochondrial activity may be an important source of anoxic injury.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Cyanides; Deoxyglucose; Energy Metabolism; Hypoxia; Kidney Medulla; Malonates; Mitochondria; Oligomycins; Oxygen Consumption; Rats; Rotenone

The uptake of [125I]T3 in rat skeletal muscle was investigated by incubating intact soleus muscles with a tracer amount of [125I]T3. At 37 C [125I]T3 uptake increased asymptotically; at 60 min the muscle contained 10% of the total [125I]T3 or 0.238 +/- 0.021% per mg wet tissue. At 0 C the [125I]T3 uptake was 1/5 of that at 37 C. The specific [125I]T3 uptake, determined by subtracting the uptake in the presence of 10 microM unlabeled T3 from the total [125I]T3 uptake, attained a plateau after 60 min. Washout experiments, done by first incubating the muscle for 60 min at 37 C or 0 C with [125I]T3 and then at 0 C for 3 h with unlabeled T3, showed that 21 +/- 2% or 58 +/- 4% of the radioactivity, respectively, was released, indicating an intracellular location of the hormone after incubation at 37 C. Addition of increasing concentrations of L-T3, D-T3 and L-T4 caused a progressive inhibition of the [125I]T3 uptake; the 50% inhibitory concentrations being 400 nM, 7 microM, and more than 15 microM, respectively. Preincubation of soleus muscles with metabolic inhibitors almost completely inhibited [125I]T3 specific uptake, with oligomycin and antimycin causing 98 +/- 4% and 81 +/- 3% reduction, respectively. Monodansylcadaverine and bacitracin, inhibitors of receptor-mediated endocytosis, reduced the specific [125I]T3 uptake in a dose-dependent manner up to 67 +/- 3% and 62 +/- 2%, respectively. These results indicate the presence of a saturable, stereospecific, and energy-dependent process responsible, at least in part, for T3 uptake in rat skeletal muscle. This specific T3 uptake may be a receptor-mediated endocytosis process.

Topics: Animals; Antimycin A; Bacitracin; Biological Transport, Active; Cadaverine; Endocytosis; In Vitro Techniques; Male; Muscles; Oligomycins; Rats; Temperature; Triiodothyronine

5,5'-Dithiobis(2-nitrobenzoate) (DTNB) is reduced in mitochondrial suspensions to 5-mercapto-2-nitrobenzoate (MNB) by 3-hydroxybutyrate and isocitrate. Although most of the MNB produced is found in the suspension medium, there is also some within the particles. The amount of MNB found in these fraction varies with the DTNB concentration used and is much lower if mitochondrial glutathione (GSH) is depleted with 1-chloro-2,4-dinitrobenzene. If hydroxybutyrate is present, the reduction of DTNB is increased by ATP and oligomycin. The pellet contains only a little MNB and GSH but these are considerably elevated by antimycin and rotenone as well as by ATP and oligomycin. If isocitrate is present, the reduction of DTNB is greatly stimulated by valinomycin, triethyltin and, to a lesser extent, oligomycin. MNB in the pellet falls and GSH concentrations are unchanged. The results suggest that with hydroxybutyrate (an NAD reducing substrate), the rate of reduction of DTNB is limited by the rate of regeneration of GSH while with isocitrate (an NADP reducing substrate) it is limited by the rate of export of MNB from the matrix.

Topics: 3-Hydroxybutyric Acid; Adenosine Triphosphate; Animals; Antimycin A; Dinitrochlorobenzene; Dithionitrobenzoic Acid; Glutathione; Hydroxybutyrates; In Vitro Techniques; Isocitrates; Mitochondria, Liver; Nitrobenzoates; Oligomycins; Oxidation-Reduction; Pentachlorophenol; Rats; Rotenone; Sulfhydryl Compounds; Triethyltin Compounds; Valinomycin

Mouse ascites-tumour cells oxidizing lactate, in a modified Ringer solution, concentrated 2-aminoisobutyrate, L-methionine or 2-(methylamino)isobutyrate about 20-fold from a 0.4 mM solution in the presence of 2-3 micrograms of nigericin/mg cellular dry wt. The ionophore increased cellular [Na+] to almost 100 mM when extracellular [Na+] was about 45 mM. Either valinomycin or the two mitochondrial inhibitors oligomycin and antimycin acting together each markedly lowered the extent to which the tumour cells concentrated amino acid, from the above factor of about 20 to roughly 2-fold. Ouabain (1 mM) had a similar effect, and further raised cellular [Na+]. The sodium pump appeared to be closely involved in amino acid uptake under these conditions.

Topics: Amino Acids; Aminoisobutyric Acids; Animals; Anti-Bacterial Agents; Antimycin A; Carcinoma, Ehrlich Tumor; In Vitro Techniques; Mice; Nigericin; Oligomycins; Ouabain; Potassium; Serum Albumin; Sodium; Valinomycin