Effect of metabolic inhibitors on formed novocaine and neutral red segregation zones in frog erythrocytes

Effects of some metabolic inhibitors, as well as of biologically active compounds (diakarb, ethidium bromide and a phenanthridine alkaloid sanguinarine) on the formed novocaine and neutral red segregation zones were studied. The volume of granules diminished under the influence of a glycolytic inhibitor iodoacetate, uncouplers of oxidative phosphorylation (2,4-dinitrophenol and carbonyl cyanide trifluoromethoxyphenylhydrozone), and respiratory inhibitors (antimycin A and rotenone), as well as under the influence of cycloheximide - an inhibitor of protein synthesis. Diakarb, ethidium bromide or sanguinarine also provoked a regression of the segregation zones. It has been found that all these compounds are inhibitors of ATPase activity of the isolated segregation zones. A possible mechanism of volume decreasing in segregation zones under the influence of both the metabolic inhibitors and diakarb, ethidium bromide and sanguinarine is discussed.     

Effect of cyanide on mitochondrial membrane depolarization induced by uncouplers

In this work, it was found that the ability of common uncouplers – carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and 2,4-dinitrophenol (DNP) – to reduce membrane potential of isolated rat liver mitochondria was diminished in the presence of millimolar concentrations of the known cytochrome c oxidase inhibitor – cyanide. In the experiments, mitochondria were energized by addition of ATP in the presence of rotenone, inhibiting oxidation of endogenous substrates via respiratory complex I. Cyanide also reduced the uncoupling effect of FCCP and DNP on mitochondria energized by succinate in the presence of ferricyanide. Importantly, cyanide did not alter the protonophoric activity of FCCP and DNP in artificial bilayer lipid membranes. The causes of the effect of cyanide on the efficiency of protonophoric uncouplers in mitochondria are considered in the framework of the suggestion that conformational changes of membrane proteins could affect the state of lipids in their vicinity. In particular, changes in local microviscosity and vacuum permittivity could change the efficiency of protonophore-mediated translocation.     

The insensitivity to uncouplers of testis mitochondrial ATPase

Albumin-free testis mitochondrial ATPase activity failed to be stimulated by either 2,4-dinitrophenol (DNP) or carbonyl cyanide rho-trifluoromethoxyphenylhydrazone (FCCP). DNP scarcely enhanced the state 4 respiration and mitochondria proved to be poorly coupled. When 1% bovine serum albumin was added to the isolation medium, DNP or FCCP stimulated ATPase nearly twofold and the dose-response curves for the uncouplers on the QO2 reached a plateau at five- to sixfold. The DNP coupling index (q) also showed a 30-40% improvement. A dose-response curve for oligomycin on the rate of [gamma-32P]ATP synthesis showed a stimulation of ATP synthase activity by 10-100 ng inhibitor/mg protein, suggesting a possible blockade of "open" F0 channels. In the albumin preparation oligomycin inhibited ATP synthesis in the range 10-100 ng/mg protein. Since testis ATPase is known to be loosely bound to the membrane, an effect of albumin, improving tightness in the interaction of the F1 and the F0 sectors of the ATPase, is suggested.     

The abnormal-shaped mitochondria in thymus lymphocytes treated with inhibitors of mitochondrial energetics

The effects of uncouplers (DNP, FCCP), oligomycin, and rotenone on the energetics and mitochondrial ultrastructure in lymphocytes have been studied. We confirmed the previous observations done on Ehrlich ascites and cardiomyocyte culture cells that uncouplers and respiratory inhibitors cause the appearance of ringlike and dumbbell-like mitochondria. It is shown that this effect does not correlate with decrease in ATP concentration, changes in oxygen consumption, or condensation of the mitochondrial matrix. FCCP (2 µM) is more effective in the induction of abnormal-form mitochondria than 240 µM DNP, oligomycin, or rotenone. Combined treatment with DNP, oligomycin, and rotenone or with DNP and rotenone produces an effect as strong as 2 µm FCCP. DNP (240 µM) and FCCP (2 µM) have a similar effect on respiration and intracellular ATP, but only the latter induces condensation of the mitochondrial matrix.     

Synergistic uncoupling of spinach chloroplasts by combinations of photophosphorylation inhibitors and carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

Combinations of low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhy-drazone (FCCP) with suboptimal concentrations of Dio-9, phloridzin, ajmaline, and dihydrodiscarine B synergistically inhibited cyclic and noncyclic photophosphorylation in spinach chloroplasts but their effects on the light-triggered ATPase were additive rather than synergistic. The effect was reversed by washing and prevented by dithioerythritol and by cistein. Carbonyl cyanide m-chlorophenylhydrazone (CCP) could replace FCCP but uncouplers of other types like atebrin did not substitute for FCCP.Combinations of FCCP with the four inhibitors synergistically uncoupled ferricyanide reduction in the presence of ADP and P_i but not in their absence. The synergistic uncoupling was not observed on the light-dependent pH rise of chloroplast suspensions.Association of FCCP with any of the inhibitors completely abolished the stimulation of proton uptake or the inhibition of electron transport induced by low concentrations of ATP.This synergistic and peculiar uncoupling can not be ascribed to a modification of membrane permeability. One possible explanation is that the effect requires a conformational state of the membrane-bound coupling factor 1 (CF₁) induced by phosphorylating conditions which would facilitate the interaction of inhibitors and FCCP with the membrane.     

Carboxyatractylate inhibits the uncoupling effect of free fatty acids

The ATP/ADP-antiporter inhibitors and ADP decrease the palmitate-induced stimulation of the mitochondrial respiration in the controlled state. The degree of inhibition decreases in the order: carboxyatractylate greater than bongkrekic acid, palmitoyl-CoA, ADP greater than atractylate. GDP is ineffective. The inhibiting concentration of carboxyatractylate coincides with this arresting the state 3 respiration. Carboxyatractylate inhibition decreases when the palmitate concentration increases. Stimulation of controlled respiration by FCCP or gramicidin D at any concentration of these uncouplers is carboxyatractylate-resistant, whereas that by low concentrations of DNP is partially suppressed by carboxyatractylate. These data together with observations that palmitate does not increase H+ conductance in bilayer phospholipid membranes and in cytochrome oxidase-asolectin proteoliposomes indicate that the ATP/ADP-antiporter is somehow involved in the uncoupling by low concentrations of fatty acids (or DNP), whereas that by FCCP and gramicidin D is due to their effect on the phospholipid bilayer. It is suggested that the antiporter facilitates translocation of palmitate anion across the mitochondrial membrane.     

Selective inhibition of pyruvate and lactate metabolism in bovine epididymal spermatozoa by dinitrophenol and alpha-cyano-3-hydroxycinnamate

The disparity between the effects of the uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) on pyruvate metabolism in bovine spermatozoa has been characterized. In bovine epididymal spermatozoa metabolizing pyruvate, the uncouplers of oxidative phosphorylation, DNP (100 μm) and FCCP (0.4 or 5 μm), decreased the intracellular ATP concentration from 30 to ～10 nmol/10⁸ cells. Both uncouplers decreased, but did not abolish, sperm motility. DNP strongly inhibited pyruvate metabolism and stimulated the appearance of free carnitine from the acetylcarnitine pool. In contrast, FCCP enhanced the oxidation of pyruvate, diminished the reduction of pyruvate to lactate, and permitted the maintenance of the normal amount of acetylcarnitine. The effects of DNP and FCCP on mitochondrial pyruvate metabolism were examined in spermatozoa treated with filipin, which renders the plasma membrane permeable to small molecules. In these cells, DNP inhibited metabolism and respiration with pyruvate or lactate, but did not affect respiration supported by acetylcarnitine. Similarly, the pyruvate translocase inhibitor, α-cyano-3-hydroxycinnamate, markedly decreased the rate of metabolism of both pyruvate and lactate. With maximally inhibitory concentrations of DNP or α-cyano-3-hydroxycinnamate, the rates of pyruvate use and lactate use were the same. Metabolism of both lactate and pyruvate and production of ATP were inhibited by similar concentrations of DNP ($\text{I}{}_{50}\text{? 7}\text{μM}$). A common mitochondrial translocase for pyruvate and lactate in bovine spermatozoa is posited. This translocase is inhibited by minimally effective uncoupling concentrations of DNP.     

A study of dependence of protein synthesis in mitochondria on the transmembrane potential

Summary 1. Incorporation of [H³]leucine into the TCA insoluble fraction of rat liver mitochondria incubatedin vitro is inhibited by uncouplers of oxidative phosphorylation. The inhibition is not correlated with the activation of mitochondrial ATPase. 2. Dependence of mitochondrial protein synthesis on the transmembrane potential is manifested in a wide range of K⁺ and Mg⁺⁺ concentrations in the incubation media. 3. The inhibitory action of uncouplers shows a lag period equal to 5–7 minutes, this lag period however is not observed when the uncoupler is added to puromycin-treated mitochondria. 4. Dependence of mitochondrial protein synthesis on the transmembrane potential, which represents a property characteristic for the inner mitochondrial membrane suggests that mitochondrial ribosomes act in close contact with the mitochondrial membrane system.Abbreviations MPS mitochondrial protein synthesis - CAP chloramphenical - CCP 2,4,6-chlorocarbonyl cyanide phenylhydrazone - FCCP p-trifluoromethoxy carbonyl cyanide phenylhydrazone - PEP phosphoenolpyruvate - Pi inorganic phosphate     

Penetrating Cations Enhance Uncoupling Activity of Anionic Protonophores in Mitochondria

Protonophorous uncouplers causing a partial decrease in mitochondrial membrane potential are promising candidates for therapeutic applications. Here we showed that hydrophobic penetrating cations specifically targeted to mitochondria in a membrane potential-driven fashion increased proton-translocating activity of the anionic uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide-p-trifluorophenylhydrazone (FCCP). In planar bilayer lipid membranes (BLM) separating two compartments with different pH values, DNP-mediated diffusion potential of H⁺ ions was enhanced in the presence of dodecyltriphenylphosphonium cation (C₁₂TPP). The mitochondria-targeted penetrating cations strongly increased DNP- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-mediated steady-state current through BLM when a transmembrane electrical potential difference was applied. Carboxyfluorescein efflux from liposomes initiated by the plastoquinone-containing penetrating cation SkQ1 was inhibited by both DNP and FCCP. Formation of complexes between the cation and CCCP was observed spectophotometrically. In contrast to the less hydrophobic tetraphenylphosphonium cation (TPP), SkQ1 and C₁₂TPP promoted the uncoupling action of DNP and FCCP on isolated mitochondria. C₁₂TPP and FCCP exhibited a synergistic effect decreasing the membrane potential of mitochondria in yeast cells. The stimulating action of penetrating cations on the protonophore-mediated uncoupling is assumed to be useful for medical applications of low (non-toxic) concentrations of protonophores.     

Effect of Metabolic Inhibitors on Membrane Potential and Ion Conductance of Rat Astrocytes

1. The aim of this study was to elucidate the effect of metabolic inhibition on the membrane potential and ion conductance of rat astrocytes. The metabolic inhibitors investigated were dinitrophenol (DNP), carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), cyanide, and oligomycin.2. Primary cultures of astroglial cells from newborn rat cerebral cortex were cultivated for 13–20 days on chamber slides. The effect of metabolic inhibitors on the cellular ATP concentration was estimated from the decrease in peak chemiluminescence from the luciferin/luciferase reaction. The membrane potential and ion conductances were measured from whole-cell recordings with the patch-clamp technique.3. After 2.0 min of incubation ATP decreased from the control level to 43%with cyanide (2 mM), 58% with DNP (1 mM), 47% with FCCP (1 M), and 69% with oligomycin (10 M).4. Under normal conditions V was –74.4±1.0 mV. DNP and FCCP both caused a rapid and reversible depolarization equivalent to a shift in the I/V curve of 8.2±1.3 and 19.7±3.8 mV, respectively. DNP decreased the slope conductance (g) by 22.1% but FCCP had no significant effect on g. In contrast, neither oligomycin nor cyanide had any significant effect on the I/V curve.5. Tetraethylammonium (TEA; 10 mM) depolarized the cells by 7.1±2.0 mV but had no significant effect on g. In the presence of TEA, DNP caused a depolarization of 52.8±3.5 mV and increased g by 45.5±9.6%. The action of FCCP was not affected by the presence of TEA.6. Perfusion of the astrocytes with a Cl^– free solution inhibited the action of DNP and FCCP. Thus the depolarization was only 4.2±1.5mV in DNP and 3.7±0.3 mV in FCCP, which were significantly smaller effects than in the presence of a high intracellular [Cl^–].7. Block of tentative K_ATP channels with tolbutamide (1 mM) or Cl^– channels with Zn²⁺ (1 mM) did not inhibit the depolarization caused by DNP or FCCP.8. In conclusion, DNP and FCCP have specific effects on the plasmalemma in rat astrocytes which may be due to opening of Cl^– channels. This effect was not seen with cyanide or oligomycin and should be considered as a possible complication when DNP and FCCP are used for metabolic inhibition.     

Inhibition of mitochondrial bioenergetics: the effects on structure of mitochondria in the cell and on apoptosis

The effects of specific inhibitors of respiratory chain, F(o)F(1)ATP synthase and uncouplers of oxidative phosphorylation on survival of carcinoma HeLa cells and on the structure of mitochondria in the cells were studied. The inhibitors of respiration (piericidin, antimycin, myxothiazol), the F(1)-component of ATP synthase (aurovertin) and uncouplers (DNP, FCCP) did not affect viability of HeLa cells, apoptosis induced by TNF or staurosporin and the anti-apoptotic action of Bcl-2. Apoptosis was induced by combined action of respiratory inhibitors and uncouplers indicating possible pro-apoptotic action of reactive oxygen species (ROS) generated by mitochondria. Short-term incubation of HeLa cells with the mitochondrial inhibitors and 2-deoxyglucose followed by 24-48 h recovery resulted in massive apoptosis. Apoptosis correlated to transient (3-4 h) and limited (60-70%) depletion of ATP. More prolonged or more complete transient ATP depletion induced pronounced necrosis. The inhibitors of respiration and uncouplers caused fragmentation of tubular mitochondria and formation of small round bodies followed by swelling. These transitions were not accompanied with release of cytochrome c into the cytosol and were fully reversible. The combined effect of respiratory inhibitors and uncouplers developed more rapidly indicating possible involvement of ROS generated by mitochondria. More prolonged (48-72 h) incubation with this combination of inhibitors caused clustering and degradation of mitochondria.     

Inhibitors of the ATP/ADP antiporter suppress stimulation of mitochondrial respiration and H+ permeability by palmitate and anionic detergents

The action of ATP/ADP-antiporter inhibitors upon the uncoupling effect of palmitate, detergents and 'classical' uncouplers has been studied. The uncoupling effect was estimated by stimulation of succinate oxidation and of H+ permeability of rat liver mitochondria in the presence of oligomycin. It is shown that carboxyatractylate (CAtr) and pyridoxal 5-phosphate (PLP) suppress the uncoupling induced by palmitate and the anionic detergents SDS and cholate, but do not affect that induced by the cationic detergents CTAB, by the non-ionic detergent Triton X-100, as well as by the 'classical' uncouplers FCCP and DNP. The results are discussed in terms of a concept assuming that the ATP/ADP-antiporter facilitates the electrophoretic export of hydrophobic anions from mitochondria.     

The binding of uncouplers of oxidative phosphorylation to rat-liver mitochondria

The binding of different uncouplers of oxidative phosphorylation to rat-liver mitochondria was measured. At pH 7.2 and about 0.7 mg mitochondrial protein/ml the percentage bound of the uncoupler added was 84% for 2,3,4,5,6-pentachlorophenol (PCP), 40% for carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 35% for 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB), 4% for α′,α′-bis (hexafluoroacetonyl)acetone (1799), and less than 4% for 2,4-dinitrophenol. These percentages are constant up to amounts of uncoupler added several times the one needed for maximal uncoupling. The values found for FCCP and TTFB are in contradiction to the proposed stoichiometric interaction of uncouplers with the coupling sites of the mitochondrial membrane.From titration experiments of the rate of O₂ uptake by rat-liver mitochondria in State 4 as a function of the uncoupler concentration in the presence of albumin or of different types of liposomes the conclusion is drawn that the negative surface charge of the mitochondrial phospholipids may be an important parameter in determining the binding of anionic uncouplers to rat-liver mitochondria.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

A systematic study was made of the action of 4-acet-amido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on active Ca2+ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca2+-selective electrodes or use of tracer 45Ca2+. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS, Ca2+ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K+ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca2+ pump, exchanging one Ca2+ ion for one proton.     

Effects of Carbonyl Cyanide Phenylhydrazones on Two Mitochondrial Ion Channel Activities

The respiratory uncouplers carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) affect the activities of two mitochondrial ion channels from mouse liver. At micromolar concentrations, the phenylhydrazones block the voltage-dependent 100-pS channel, mCS, and induce the multiple-conductance-level channel, MCC. The binding site(s) involved in perturbation of channel activities are probably distinct from the sites involved in uncoupling of oxidative phosphorylation which occurs at nanomolar concentrations of the phenylhydrazones. The effects of FCCP and CCCP on the mitochondrial ion channels could be partially reversed by washing with fresh media and were always reversed by perfusion with dithiothreitol. These results indicate that the effects of the phenylhydrazones on mitochondrial ion channels may be related to the ability of these compounds to act as sulfhydryl reagents and not to their protonophoric and uncoupling activity.     

Secretion in yeast: translocation and glycosylation of prepro-alpha-factor in vitro can occur via an ATP-dependent post-translational mechanism

In an in vitro system comprising a yeast cell-free translation system, yeast microsomes and mRNA encoding prepro-alpha-factor, the translocation of this protein across the membrane of the microsomal vesicle and its glycosylation could b uncoupled from its translation. Such post-translational processing is dependent upon the presence of ATP in the system. It is not, however, affected by a variety of uncouplers, ionophores or inhibitors, including carbonyl cyanide m-chlorophenyl hydrazone (CCCP), valinomycin, nigericin, dinitrophenol (DNP), potassium cyanide (KCN) or N-ethyl maleimide (NEM). This mechanism of translocation is significant as it indicates that a protein of 18 000 daltons is capable of crossing an endoplasmic reticulum-derived membrane post-translationally. For the moment, this phenomenon seems to be restricted to prepro-alpha-factor in the yeast in vitro system. Neither invertase nor IgG chi light chain could be translocated post-translationally in yeast, nor was such processing observed for prepro-alpha-factor in a wheat germ system supplemented with canine pancreatic microsomes.     

Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization

Mitochondria in plant cells undergo fusion and fission frequently. Although the mechanisms and proteins of mitochondrial fusion are well known in yeast and mammalian cells, they remain poorly understood in plant cells. To clarify the physiological requirements for plant mitochondrial fusion, we investigated the fusion frequency of mitochondria in tobacco cultured cells using the photoconvertible fluorescent protein Kaede and some physiological inhibitors. The latter included two uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of mitochondrial ATP synthase, oligomycin, and an actin polymerization inhibitor, latrunculin B (Lat B). The frequency of mitochondrial fusion was clearly reduced by DNP, CCCP and oligomycin, but not by Lat B, although Lat B severely inhibited mitochondrial movement. Moreover, DNP, CCCP and oligomycin evidently lowered the cellular ATP levels. These results indicate that plant mitochondrial fusion depends on the cellular ATP level, but not on actin polymerization.     

Reconstitution of cytochrome oxidase vesicles and conferral of sensitivity to energy transfer inhibitors

Summary 1. Phospholipids and cytochrome oxidase solubilized with chotate were reconstituted either by dialysis or by dilution of the detergent. The reconstituted cytochrome oxidase vesicles oxidized ascorbate-cytochromec at a rate which was low, insensitive to energy transfer inhibitors and markedly stimulated by uncouplers of oxidative phosphorylation. The rate of reconstitution was dependent on pH, on the concentration of cholate and on the presence of high concentrations of monovalent ions or low concentrations of divalent ions. The integrity of the cytochrome oxidase vesicles was retained after freeze-drying, provided sucrose was present during the process. 2. Reconstitution with pure phospholipids revealed that cardiolipin was required for the marked stimulation of respiration by uncouplers. 3. Cytochrome oxidase vesicles were reconstituted in the presence of hydrophobic mitochondrial proteins which contained oligomycin-sensitive ATPase. The resulting vesicles oxidized ascorbate-cytochromec at a rapid rate which was not enhanced by uncouplers. Addition of an energy transfer inhibitor such as rutamycin resulted in a partial inhibition of respiration which was released by uncouplers. 4. Cytochrome oxidase vesicles reconstituted in the presence of phenol red were rather impermeable to protons and became very permeable on addition of uncouplers. When the reconstitution was performed in the presence of the hydrophobic proteins from mitochondria, proton translocation became partially sensitive to rutamycin. 5. These observations are consistent with some of the formulations of the chemiosmotic hypothesis.     

Role of dark respiration in photoinhibition of photosynthesis and its reactivation in the cyanobacterium Anacystis nidulans

Photoinhibition of photosynthesis and its reactivation was studied in the cyanobaterium A. nidulans in the presence of the respiratory inhibitor sodium azide, the uncouplers carbonyl cyanide p -(trifluoromethoxy)-phenylhydrazone (FCCP) and carbonyl cyanide m -chlorophenylhydrazone (CCCP) and the photosystem I elicitor phenazine methosulphate (PMS). Inhibition of dark respiration by azide increased the susceptibility of the cyanobacterium to photoinhibition. Both FCCP and CCCP also remarkably affected the process of photoinhibition in A. nidulans. The PMS at lower photoinhibitory light intensity partially protected A. nidulans from photoinhibition. The recovery from photoinhibition in the presence of azide or FCCP was slow and normal photosynthesis could not be resumed even after a longer period of incubation under suitable reactivating condition. Thus dark respiration has a key function in the process of photoinhibition of photosynthesis and its reactivation in the cyanobacterium A. nidulans.