The impairing effects of chaetochromin D on mitochondrial respiration and structure

Chaetochromin D, a toxic secondary metabolite ofChaetomlum graclle, was examined for impairing effects on mitochondrial respiration and structure (swelling-induction) using isolated rat liver mitochondria to gain Insight into the molecular mechanism for Its cytotoxicity. Chaetochromin D exerted similar mode of effects to those of chaetochromin A, cephalochromin, and ustilaginoldin A on mitochondrial reactions, causing uncoupling of oxidative phosphorylation, depression of state 3 respiration, and induction of drastic swelling In mitochondria. Chaetochromin D induced the same style of swelling as that induced by chaetochromin A, being characterized by a very high rate and small amplitude of swelling. The swelling terminated In the middle and the amplitude was about half of the full swelling. Once the quick swelling ceased in the middle, subsequent swelling could not be elicited by the second addition of chaetochromin D at any of the concentrations tested.     

Nature of proton cycling during gramicidin uncoupling of oxidative phosphorylation

Addition of gramicidin D to liver mitochondria, incubated in low- or high-salt media, results in stimulation of respiration in the absence or presence of depression of delta muH, respectively. Gramicidin D concentrations 2 orders of magnitude higher are required in the low-salt media with full uncoupling at 1 nmol of gramicidin.mg-1. The stimulation of respiration is not accompanied by increased passive proton influx in low-salt media. In high-salt media, the extent of respiratory stimulation and the extent of delta muH depression differ according to the nature and concentration of cation. The flow-force relationship is very steep when gramicidin D induced uncoupling occurs in low-salt media and much less steep in high-salt media. A multiplicity of flow-force relationship, respiratory rate vs delta muH, is obtained, the slope of which depends on the nature and concentration of cation, and which can be reproduced by computer simulation by introducing a variable extent of proton cycling either in the membrane or in the pump. The apparent proton conductance, as analyzed in the relationship of Je/delta muH vs delta muH, increases in the so-called ohmic and nonohmic regions according to whether gramicidin D is added in high-salt or low-salt media, respectively. Titration with antimycin of the respiratory control ratio (RCR) in gramicidin D treated mitochondria leads to a depression of the RCR in high-salt but not in low-salt media. The view is discussed that in low-salt media the gramicidin D induced uncoupling is due to a cycling of protons within a proton domain operationally located at or near the proton pump.(ABSTRACT TRUNCATED AT 250 WORDS)     

The action of valinomycin in uncoupling corn mitochondria

Valinomycin in the presence of potassium is a potent uncoupler of corn (Zea mays L.) mitochondria, eliminating respiratory control. Valinomycin produces higher steady state potassium phosphate swelling which can be reversed to give active shrinkage if mersalyl is added to block the Pi⁻/OH⁻ antiporter. Respiration declines concurrently. Uncouplers accelerate the shrinkage and restore the respiration. The same results can be obtained with sodium phosphate if gramicidin D is substituted as ionophore.     

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.     

The ATP/ADP-antiporter is involved in the uncoupling effect of fatty acids on mitochondria

The ATP/ADP-antiporter inhibitors and the substrate ADP suppress the uncoupling effect induced by low (10-20 microM) concentrations of palmitate in mitochondria from skeletal muscle and liver. The inhibitors and ADP are found to (a) inhibit the palmitate-stimulated respiration in the controlled state and (b) increase the membrane potential lowered by palmitate. The degree of efficiency decreases in the order: carboxyatractylate (CAtr) greater than ADP greater than bongkrekic acid, atractylate. GDP is ineffective, Mg.ADP is of much smaller effect, whereas ATP is effective at much higher concentration than is ADP. Inhibitor concentrations, which maximally suppress the palmitate-stimulated respiration, correspond to those needed for arresting the state 3 respiration. The extent of the CAtr-sensitive stimulation of respiration by palmitate has been found to decrease with an increase in palmitate concentration. Stimulation of the controlled respiration by p-trifluoromethoxycarbonylcyanide phenylhydrozone (FCCP) and gramicidin D at any concentrations of these uncouplers is CAtr-insensitive, whereas that caused by a low concentrations of 2,4-dinitrophenol and dodecyl sulfate is inhibited by CAtr. The above effect of palmitate develops immediately after addition of the fatty acid. It is resistant to EGTA as well as to inhibitors of phospholipase (nupercain) and of lipid peroxidation (ionol). Moreover, palmitate accelerates spontaneous release of the respiratory control, developing in rat liver mitochondria under certain conditions. This effect takes several minutes, being sensitive to EGTA, nupercain and ionol. Like the fast uncoupling, this slow effect is inhibited by ADP but CAtr and atractylate are stimulatory rather than inhibitory. In artificial planar phospholipid membrane, palmitate does not increase the membrane conductance, FCCP increases it strongly and dinitrophenol only slightly. In cytochrome oxidase proteoliposomes, FCCP, gramicidin and dinitrophenol (less effectively) lower, whereas palmitate enhances the cytochrome-oxidase-generated membrane potential. In this system, monensin substitutes for palmitate. It is concluded that the ATP/ADP antiporter is somehow involved in the uncoupling effect caused by low concentrations of palmitate and, partially, of dinitrophenol, whereas uncoupling produced by FCCP and gramicidin is due to their action on the phospholipid part of the mitochondrial membrane. A possible mechanism of this effect is discussed.     

Dependence of Bacillus subtilis cell respiration on monovalent cations]

Nigericin, monensin, valinomycin + carbonyl-cyanide-m-chlorophenylhydrazone and gramicidin inhibit the respiration of Bacillus subtilis cells incubated with NAD-dependent substrates or succinate, but not with ascorbate + N,N,N',N'-tetramethyl-p- phenylene-diamine. The level of inhibition was decreased by potassium ions and, in a lower degree, by sodium or ammonium ions. The results obtained suggest that the respiration of Bacillus subtilis depends on the presence of monovalent cations whose effects seem to be directed at complexes I, III and probably complex II of the respiratory chain.     

A Comparison of Mitochondria Isolated from Male-Sterile and Nonserile Cytoplasm Etiolated Corn Seedlings

Toxins from Helminthosporium maydis race T and Phyllosticta maydis have been found to affect the functional processes of corn mitochondria isolated from Texas male-sterile (T) cytoplasm, but not of mitochondria isolated from nonsterile (N) cytoplasm. The effects of chemicals known to induce responses similar to those of the toxin were compared on mitochondria isolated from T and N cytoplasm inbreds (W64A, Zea mays L.). Valinomycin, gramicidin, and decenylsuccinic acid (DSA) each caused more swelling (measured by transmission changes in %) of N mitochondria than of T mitochondira. The stimulation of exogenous NADH oxidation was the same for N and T mitochondria in the valinomycin, DSA, and Ca²⁺ plus phosphate treatments, was greater for T mitochondria than for N mitochondria in the gramicidin and DNP treatments, and was greater for N mitochondrai than for T mitochondira in the Ca⁺² minus phosphate treatment. Sodium azide inhibited NADH oxidation equally for N and T mitochondria. In addition, N and T mitochondria had similar respiration rates for various substrates and equal efficiencies of oxidative phosphorylation. In contrast to the specificity of toxins for T mitochondria, none of the treatment effects were specific for N or T mitochondria. The results indicate that mitochondria isolated from N and T cytoplasm generally respond similarly to various conditions, but that there can be quantitative differences in the response. The extent to which these differences represent cytoplasmically controlled modification of mitochondrial physiology or structure is not known.     

Effect of inductors of alkali cation permeability on cytochrome c bound to mitochondrial membrane]

The effect of inductors of alkali cation permeability--valinomycin, gramicidin A, gramicidin S and its N,N'-diacetyl derivative--on rat liver mitochondria during respiration has been studied. It is shown that valinomycin, gramycidin A and diacetylgramicidin S at optimal concentration for uncoupling cause two-phase activation of mitochondrial respiration and that this effect results from cytochrome c solubilization. Gramicidin S at optimal concentration cannot remove cytochrome c from the respirating mitochondria. It is suggested that this property of gramicidin S is owned to cytochrome c immobilisation in membrane, due to the effect of this compound.     

Gramicidin D and dithiothreitol effects on erythrocyte exovesiculation

The use of either diphenylhexatriene, trimethylamino-diphenylhexatriene, or heptadecyl-hydroxycoumarin (C17-HC) allows, simultaneously and with the same molecule, the induction of erythrocyte exovesiculation and labeling of the released vesicles with the fluorescent probe. This method was used to evaluate gramicidin D (a channel-forming peptide) and dithiothreitol (a reducing agent) effects on the human erythrocytes vesiculation process. The release of cholesterol and phospholipids in exovesicles at longer incubation times was only detectable in the presence of gramicidin or dithiothreitol. When C17-HC was used to induce the vesiculation, the presence of gramicidin or dithiothreitol lead to a drastic decrease on the [phospholipids]/[cholesterol] ratio. However, in the samples with dithiothreitol, this variation did not result in the expectable decrease of membrane fluidity. These effects can be related with the presence of lipid rafts, the transbilayer lipids reorientation induced by gramicidin or dithiothreitol, and the cholesterol-dependent gramicidin channels inactivation.     

Effects of hydroxylated polychlorinated biphenyls on mouse liver mitochondrial oxidative phosphorylation.

The effects of three tetrachlorobiphenylols [2',3',4',5'-tetrachloro-2-biphenylol (1); 2',3',4',5'-tetrachloro-4- biphenylol (2); and 2',3',4',5'-tetrachloro-3-biphenylol (3)]; three monochlorobiphenylols [5-chloro-2-biphenylol (5), 3-chloro-2-biphenylol (6); and 2-chloro-4-biphenylol (7)] and a tetrachlorobiphenyldiol [3,3',5,5'-tetrachloro-4,4'-biphenyldiol (4) on respiration, adenosine triphosphatase (ATPase) activity, and swelling in isolated mouse liver mitochondria have been investigated. Tetrachlorobiphenylols (1-3) and the tetrachlorobiphenyldiol (4) inhibited state-3 respiration in a concentration-dependent manner with succinate as substrate (flavin adenine dinucleotide [FAD]-linked) and the tetrachlorobiphenyldiol (4) caused a more pronounced inhibitory effect on state-3 respiration than the other congeners. The monochlorobiphenylols 5-7 were less active as inhibitors of state-3 mitochondrial respiration and significant effects were observed only at higher concentration (greater than or equal to 0.4 microM). However, in the presence of the nicotinamide adenine dinucleotide (NAD)-linked substrates (glutamate plus malate), hydroxylated PCBs (1-7) significantly inhibited mitochondrial state-3 respiration in a concentration-dependent manner. Compounds 5, 6, and 7 uncoupled mitochondrial oxidative phosphorylation only in the presence of FAD-linked substrate as evidenced by increased oxygen consumption during state-4 respiratory transition, stimulating ATPase activity, releasing oligomycin-inhibited respiration, and inducing mitochondrial swelling (5, 6, and 7). Tetrachlorobiphenylols 1, 2, and 3 had no effect on mitochondrial ATPase activity while the tetrachlorobiphenyldiol, 4, decreased the enzyme activity. The possible inhibitory site of electron transport by these compounds and their toxicologic significance is discussed.     

Change in lipid-protein interactions in the membranes of bacteria exposed to gramicidin S]

Effect of cyclopeptide antibiotic gramicidin S on some enzymes and physical state of isolated Micrococcus lysodeikticus membranes is studied. Malate and lactate dehydrogenases were monotonously inhibited under the increase of gramicidin S concentration, while the activity of NADH-dehydrogenase firstly decreased and then reversed to the initial level under further increase of gramicidin S concentration. The oxygen uptake under oxidation of NADH and malate with membranes almost completely inhibited by the antibiotic, while the activity of ascorbate-TMPD-oxidase activity slightly inhibited by the same concentration of gramicidin. The addition of Triton X-100 completely eliminated the inhibitory effect of gramicidin on malate dehydrogenase. The introduction into the membrane of spine probes (2,2,6,6-tetramethyl-4-palmitoylamidopiperidine-1-oxile and 2(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxyazolidinyloxile) revealed that gramicidin caused the condensation of membrane lipid component. It is suggested that ionic interaction of gramicidin S with membrane phospholipids brings to "a freezing" of lipids which is a direct cause of impairing the activity of membrane respiration enzymes and the change of their position in the lipid matrix, thus inhibiting energy-producing processes in cell.     

Localization of gramicidin S on the cytoplasmic membrane of Bacillus brevis and its effect on the activity of membrane enzymes

It was shown that malate dehydrogenase of isolated membranes of the gramicidin S producer Bacillus brevis var. G.-B. (R.-form) is completely inhibited by the antibiotic (approximately 200 mkg/mg of protein). Succinate and NADH dehydrogenases at concentration up to 1 mg per mg of protein are insensitive to it, while corresponding oxidases are inhibited by the antibiotic not more than by 65 -- 75% apparently due to partial damage of the terminal parts of the respiratory chain. The respiration of the producer intact cells is inhibited by exogenous gramicidin S by not more than 55 -- 60%, while the respiration of antibiotic-sensitive cells of M.lysodeikticus is inhibited completely. It was shown that phosphatidyl ethanolamine (50%), phosphatidyl glycerol (15% and diphosphatidyl glycerol (25%) are the major phospholipid components of the membranes of the given strain of Bac. brevis. It was assumed that the resistance of Bac. brevis cells to gramicidin S is partly due to the constant ratio of the charged and amphoteric phospholipids. Using 31P-NMR spectroscopy, the kinetics of free phosphoric compounds in the cells and cell extracts of Bac. brevis during culture growth and gramicidin S synthesis were studied. The content of carbohydrate monophosphate, remained unaffected, while that of nucleoside di- and triphosphates and dinucleotides was low and at definite density and gramicidin S content (above 100 mkg/ml) fell down below the resolution capacity of the method employed. Evidence for gramicidin S localization of the Bac. brevis membrane and possible causes for the manifestation of the NADH dehydrogenase activity at a certain stage of culture growth are discussed.     

A preliminary scanning tunnelling microscopy study of the surface-organised structures of gramicidin S hydrochloride on highly oriented pyrolytic graphite

For an initial study of potentially surface-structural self-organising systems of biological significance by scanning tunnelling microscopy (STM), gramicidin S, a pseudocentrosymmetric cyclodecapeptide with antibiotic properties, was chosen as prototype, recognising its structure as having both intramolecular and intermolecular hydrogen-bond forming propensity. The surface-organised structures, based on gramicidin S hydrochloride deposited on a highly oriented pyrolytic graphite (HOPG) substrate, have been observed by STM in air under ambient conditions. These are characterised in the main by rectangular or rectangle-like structural elements identified with the individual gramicidin S hydrochloride molecules. Two kinds of arrangements of gramicidin S hydrochloride in a two-dimensional array are found, i.e., as a centred rectangular lattice and a primitive rectangular lattice. The STM topographical arrays and the molecular dimensions obtained are in good quantitative agreement with the corresponding X-ray crystallographic data. The differences between the STM results, the theoretical models, and the X-ray crystallographic data are attributed to the intermolecular interactions present in the three-dimensional gramicidin S crystal but absent in the lower dimensional arrays and to the environments in which a gramicidin S hydrochloride molecule finds itself during deposition and drying on the HOPG substrate.     

Effects of Fluoride on Mitochondrial Activity in Higher Plants

The effects of fluoride on respiration of plant tissue and mitochondria were investigated. Fumigation of young soybean plants (Glycine max Merr. cv. Hawkeye) with 9–12 μg × m^?3 HF caused a stimulation of respiration at about 2 days of treatment followed by inhibition 2 days later. Mitochondria isolated from the stimulated tissue had higher respiration rates, greater ATPase activity, and lower P/O ratios, while in mitochondria from inhibited tissue, all three were reduced. Treatment of etiolated soybean hypocotyl sections in Hoagland's solution containing KF for 3 to 10 h only resulted in inhibition of respiration. Mitochondria isolated from this tissue elicited increased respiration rates with malate as substrate and inhibited respiration with succinate. With both substrates respiratory control and ADP/O ratios were decreased. Direct treatment of mitochondria from the etiolated soybean hypocotyl tissue with fluoride resulted in inhibition of state 3 respiration and lower ADP/O ratios with the substrates succinate, malate, and NADH. Fluoride was also found to increase the amount of osmotically induced swelling and cause a more rapid leakage of protein with mitochondria isolated from etiolated corn shoots (Zea mays L. cv. Golden Cross Bantam). The results are discussed with respect to possible effects of fluoride on mitochondrial membranes.     

Inhibition by secalonic acid D of oxidative phosphorylation and Ca2+-induced swelling in mitochondria isolated from rat livers

The in vitro biological activity of secalonic acid D, a mycotoxin from Aspergillus ochraceus, was studied to assess its cytotoxicity for isolated rat liver mitochondria. Secalonic acid D uncoupled the oxidative phosphorylation of mitochondria and caused a mild inhibition of state 3 respiration. Secalonic acid D weakly enhanced latent ATPase activity in mitochondria but suppressed 2,4-dinitrophenol-stimulated ATPase activity. Secalonic acid D did not induce pseudoenergized swelling of mitochondria and markedly inhibited the Ca2+-induced swelling of mitochondria in KCl isotonic solution.     

Inhibition by secalonic acid D of oxidative phosphorylation and Ca2+-induced swelling in mitochondria isolated from rat livers.

The in vitro biological activity of secalonic acid D, a mycotoxin from Aspergillus ochraceus, was studied to assess its cytotoxicity for isolated rat liver mitochondria. Secalonic acid D uncoupled the oxidative phosphorylation of mitochondria and caused a mild inhibition of state 3 respiration. Secalonic acid D weakly enhanced latent ATPase activity in mitochondria but suppressed 2,4-dinitrophenol-stimulated ATPase activity. Secalonic acid D did not induce pseudoenergized swelling of mitochondria and markedly inhibited the Ca2+-induced swelling of mitochondria in KCl isotonic solution.     

Water regulated effects of photosynthetic substrate supply on soil respiration in a semiarid steppe

Soil respiration is an important part of the global carbon (C) cycle and the largest component of C flux from terrestrial ecosystems to the atmosphere. Here, we investigated possible effects of photosynthetic substrate supply on soil respiration in a semiarid ecosystem. A field experiment combining water addition and shading (low and high shading) treatments was conducted to manipulate photosynthetic substrate supply in a temperate semiarid steppe in two growing seasons. Our result showed that water addition and/or low shading significantly increased net primary productivity (ecosystem‐level photosynthetic substrate supply) and soil respiration in both two growing seasons. However, the effects of high shading on net primary productivity and soil respiration depended on soil water condition, which were negative in wet year (2008) but positive in dry year (2009). On the diel timescale, soil respiration was out of phase with soil temperature and leaf net photosynthesis, but in phase with leaf sugar and starch contents (leaf‐level photosynthetic substrate production). The results indicated that photosynthetic substrate supply was an important factor in regulating soil respiration on both daily and seasonal timescales. Moreover, its effect on soil respiration increased with increasing water availability in this region. The predominant role of C assimilate supply on soil respiration indicates that the predicted positive influence of rising temperature on soil respiration will be simultaneously mediated by substrate supply and water availability in semiarid steppe ecosystems.     

The uncoupling effect of the nonsteroidal anti-inflammatory drug nimesulide in liver mitochondria from adjuvant-induced arthritic rats

The aim of the present study was to evaluate the changes caused by adjuvant-induced arthritis in liver mitochondria and to investigate the effects of the nonsteroidal anti-inflammatory drug nimesulide. The main alterations observed in liver mitochondria from arthritic rats were: higher rates of state IV and state III respiration with beta-hydroxybutyrate as substrate; reduced respiratory control ratio and impaired capacity for swelling dependent on beta-hydroxybutyrate oxidation. No alterations were found in the activities of NADH oxidase and ATPase. Nimesulide produced: (1) stimulation of state IV respiration; (2) decrease in the ADP/O ratio and in the respiratory control ratio; (3) stimulation of ATPase activity of intact mitochondria; (4) inhibition of swelling driven by the oxidation of beta-hydroxybutyrate; (5) induction of passive swelling due to NH(3)/NH(4)+ redistribution. The activity of NADH oxidase was insensitive to nimesulide. Mitochondria from arthritic rats showed higher sensitivity to nimesulide regarding respiratory activity. The results of this work allow us to conclude that adjuvant-induced arthritis leads to quantitative changes in some mitochondrial functions and in the sensitivity to nimesulide. Direct evidence that nimesulide acts as an uncoupler was also presented. Since nimesulide was active in liver mitochondria at therapeutic levels, the impairment of energy metabolism could lead to disturbances in the liver responses to inflammation, a fact that should be considered in therapeutic intervention.     

Effect of streptavidins with varying biotin binding affinities on the properties of biotinylated gramicidin channels

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.     

The effect of changes in gramicidin conformation on bilayer lipid properties.

The effects of two different gramicidin conformations on lipid phase behaviour and dynamics are compared. Samples of chain-perdeuterated dimyristoylphosphatidylcholine containing gramicidin were first prepared with gramicidin in a state having a circular dichroism spectrum generally identified as corresponding to the non-channel conformation. The effects, on bilayer lipid properties, of gramicidin in this conformation were then determined using deuterium nuclear magnetic resonance measurements of acyl chain orientational order and transverse relaxation times as a function of temperature. These samples were then incubated at 65 degrees C to convert the gramicidin to a state with a circular dichroism spectrum of the type generally identified with the channel conformation. The nuclear magnetic resonance measurements were then repeated. In the gel phase, it was found that transverse relaxation time and chain orientational order of the lipid were insensitive to gramicidin conformation. In the liquid crystalline phase, gramicidin in the channel conformation was found to have a slightly larger effect on transverse relaxation and orientational order than gramicidin in the non-channel conformation. The perturbation of the phase behavior by gramicidin was found to be relatively insensitive to gramicidin conformation.