Involvement of Reactive Oxygen Species in Plant Mitochondrial Response to Low-Temperature Stress

The review presents data for the generation of the reactive oxygen species (ROS) in mitochondria during low-temperature stress. The relation of lipid peroxidation processes in membranes to the induction of low-temperature stress is demonstrated. The data for the involvement of the mitochondrial uncoupling proteins and the processes of uncoupling oxidation and phosphorylation in the regulation of mitochondrial ROS formation during low-temperature stress are considered.     

Properties of substituted 2-trifluoromethylbenzimidazoles as uncouplers of oxidative phosphorylation

1. The activity of 25 substituted 2-trifluoromethylbenzimidazoles in uncoupling oxidative phosphorylation by rat-liver mitochondria has been compared. 2. For halogen- or mixed-halogen- and alkyl-substituted analogues, uncoupling activity was proportional to the acidity of the imidazole -NH group. Tetrachloro-2-trifluoromethylbenzimidazole was the most active (50% uncoupling of oxidative phosphorylation at 7.9x10(-8)m, pK5.04). Nitro-substituted analogues were less active than predicted from pK considerations or from partition-coefficient measurements. 3. Introduction of an -NH(2) or -CO(2)H substitutent caused a loss of uncoupling activity, as did alkylation at position 1 of the imidazole ring. 4. Benzimidazoles active as uncouplers stimulated mitochondrial adenosine triphosphatase but not all stimulated the oxidation of succinate in the absence of a phosphate acceptor. 5. 4,5-Dichloro-2-trifluoromethylbenzimidazole inhibited the succinate-oxidase system at about the same concentration required for uncoupling (0.52mum for 50% inhibition of both activities) and the site of this inhibition appears to lie between succinate dehydrogenase and cytochrome b.     

Cold-Shock 310-kD Protein Uncouples Oxidative Phosphorylation in Plant Mitochondria

We studied the effects of cold-shock 310-kD protein (CSP310) isolated from winter rye seedlings on the energetic activity of plant mitochondria. CSP310 was shown to enhance nonphosphorylating respiration and uncoupled oxidative phosphorylation in isolated mitochondria. The uncoupling effect was enhanced with increasing protein concentration. An antibody against CSP310 interfered with the uncoupling effect of CSP310. Free fatty acids were not evidently involved in uncoupling. The physiological role of uncoupling between oxidation and phosphorylation during plant adaptation to low temperatures is discussed.     

Mitochondrial protonophoric activity induced by a thyromimetic fatty acid analogue

Calcium-dependent uncoupling of liver mitochondrial oxidative phosphorylation by a non-metabolizable long chain fatty acyl analogue was compared with uncoupling induced by in vivo thyroid hormone treatment. beta,beta'-Methyl-substituted hexadecane alpha, omega-dioic acid (Medica 16) is reported here to induce a saturable 20-30% decrease in liver mitochondrial DeltaPsi, DeltapH and protonmotive force which proceeds in the presence of added Ca(2+) to cyclosporin A-sensitive mitochondrial permeabilization. Ca(2+)-dependent uncoupling by Medica 16 was accompanied by atractylate-enhanced, bongkrekic-inhibited activation of mitochondrial Ca(2+) efflux. The direct mitochondrial effect exerted in vitro by Medica 16 is similar to that induced by in vivo thyroid hormone treatment. Hence, the thyromimetic protonophoric activity of Medica 16 and the uncoupling activity of TH converge onto components of the mitochondrial permeabilization transition pore.     

Participation of ADP/ATP- and Aspartate/Glutamate Antiporters in the Uncoupling Action of Fatty Acids in Liver Mitochondria of the Frog Rana temporaria

Data are presented on molecular mechanisms of uncoupling of oxidative phosphorylation by fatty acids (laurate) in liver mitochondria of one of the poikilothermal animals, the frog Rana temporaria. It has been shown that the uncoupling action of laurate in frog liver mitochondria, like in those of mammals, occurs with participation of protein carriers of anions of the inner mitochondrial membrane, ADP/ATP- and aspartate/glutamate antiporters. At the same time, in frog liver mitochondria the uncoupling activity of laurate is lower than in liver mitochondria of mammals (white mice). Seasonal differences in the laurate uncoupling activity in frog liver mitochondria are revealed: it is much lower in April, than in January, the season of metabolic depression. This difference is due to that in January the degree of participation of the aspartate/glutamate antiporter in the uncoupling is considerably decreased.     

Uncoupling effect of anacardic acids from cashew nut shell oil on oxidative phosphorylation of rat liver mitochondria

Anacardic acids are one of natural products found in not only the cashew nut shell oil but also the nut and fruit juice. The present study was conducted to investigate the uncoupling effect of anacardic acids on oxidative phosphorylation of rat liver mitochondria using succinate (plus rotenone) as a substrate. Four anacardic acids with C15:0, C15:1, C15:2 or C15:3 as an alkyl side chain exhibited uncoupling effects similar to the classical uncoupler, 2,4-dinitrophenol on ADP/O ratio, state 4 and respiratory control ratio (RCR). Anacardic acid with C15:1 side chain was most effective for uncoupling of these compounds. Salicylic acid, which has no alkyl side chain, exhibited a very weak uncoupling effect on oxidative phosphorylation. When the carboxyl group in anacardic acids was lost converting them to the corresponding cardanols, uncoupling activity dramatically decreased regardless of the number of double bonds in the long alkyl chain. These results suggest that the C15 alkyl side chain as well as the carboxyl group may play an important role in assisting the uncoupling activity of anacardic acids in liver mitochondria of animals. This study provides the first evidence of an uncoupling effect of anacardic acids on liver mitochondria     

Uncoupling action of antibiotic sporaviridins with rat-liver mitochondria.

The effects of the glycoside antibiotic sporaviridins (SVDs) on oxidative phosphorylation of rat-liver mitochondria were examined. SVDs released state 4 respiration, dissipated transmembrane electrical potential, and accelerated ATPase activity. These facts demonstrated that SVDs are potent uncouplers of oxidative phosphorylation. During the uncoupling caused by SVDs, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, suggesting that SVDs greatly enhanced nonspecific permeability of the inner mitochondrial membrane. It is suggested that the uncoupling action of SVDs might be caused by dissipation of proton electrochemical potential due to an increase in the permeability of inner mitochondrial membrane.     

Uncoupling Action of Antibiotic Sporaviridins with Rat-liver Mitochondria

The effects of the glycoside antibiotic sporaviridins (SVDs) on oxidative phosphorylation of rat-liver mitochondria were examined. SVDs released state 4 respiration, dissipated transmembrane electrical potential, and accelerated ATPase activity. These facts demonstrated that SVDs are potent uncouplers of oxidative phosphorylation. During the uncoupling caused by SVDs, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, suggesting that SVDs greatly enhanced nonspecific permeability of the inner mitochondrial membrane. It is suggested that the uncoupling action of SVDs might be caused by dissipation of proton electrochemical potential due to an increase in the permeability of inner mitochondrial membrane.     

Inhibition and uncoupling of photophosphorylation in isolated chloroplasts by organotin, organomercury and diphenyleneiodonium compounds

1. Trialkyltin, triphenyltin and diphenyleneiodonium compounds inhibited ADP-stimulated O(2) evolution by isolated pea chloroplasts in the presence of phosphate or arsenate. Tributyltin and triphenyltin were the most effective inhibitors, which suggests a highly hydrophobic site of action. Phenylmercuric acetate was a poor inhibitor of photophosphorylation, which suggests that thiol groups are not involved. 2. Triethyltin was a potent uncoupler of photophosphorylation by isolated chloroplasts in media containing Cl(-), but had little uncoupling activity when Cl(-) was replaced by NO(3) (-) or SO(4) (2-), which are inactive in the anion-hydroxide exchange. It is suggested that uncoupling by triethyltin is a result of the Cl(-)-OH(-) exchange together with a natural uniport of Cl(-). Tributyltin, triphenyltin and phenylmercuric acetate had low uncoupling activity, probably because in these compounds the uncoupling activity is partially masked by inhibitory effects. 3. At high concentrations the organotin compounds caused inhibition of electron transport uncoupled by carbonyl cyanide m-chlorophenylhydrazone or NH(4)Cl. At these high concentrations the organotin compounds may be producing a detergent-like disorganization of the membrane structure. In contrast, diphenyleneiodonium sulphate inhibited uncoupled electron transport at low concentrations; however, this inhibition is less than the inhibition of photophosphorylation, which suggests that the compound also inhibits the phosphorylation reactions as well as electron transport. 4. The effects of these compounds on basal electron transport were complex and depended on the pH of the reaction media. However, they can be explained on the basis of three actions: inhibition of the phosphorylation reactions, uncoupling and direct inhibition of electron transport. 5. The inhibition of cyclic photophosphorylation in the presence of phenazine methosulphate by diphenyleneiodonium sulphate shows that it inhibits in the region of photosystem 1.     

Interaction of free fatty acids with mitochondria during uncoupling of oxidative phosphorylation

The activity of free saturated fatty acids (caprylic, capric, lauric, myristic, palmitic and stearic) as inducers and regulators of uncoupling of oxidative phosphorylation with participation of ADP/ATP antiporter, aspartate/glutamate antiporter and cyclosporin A-sensitive structure was investigated in experiments on rat liver mitochondria. It is established that at equal uncoupling activity of fatty acids the regulatory effect is minimal for caprylic acid and raised with increasing the hydrophobicity of fatty acids reaching the maximum value for stearic acid. There exists the linear dependence of the regulatory effect value of fatty acids on fatty acids content in the hydrophobic region of the inner membrane. The model that describes the interaction of fatty acids with the hydrophobic region of the mitochondrial inner membrane preserving functional activity of organelles is developed. It is established that if molecules of various fatty acids being in the hydrophobic region of the membrane are equally effective as uncoupling regulators, their specific uncoupling activity is different. Caprylic acid, a short-chain fatty acid, possesses the highest uncoupling activity. As the acyl chain length increases, the specific uncoupling activity of fatty acids reduces exponentially. Under these conditions components of the uncoupling activity sensitive to glutamate and carboxyatractylate and glutamate and insensitive to these reagents (but sensitive to cyclosporin A) change approximately equally.     

Uncoupling effect of protonophoric and nonprotonophoric analogs of carbonyl cyanide phenylhydrazone on mitochondrial oxidative phosphorylation

Analogs of carbonyl cyanide phenylhydrazone providing no reaction with nucleophilic groups and lacking acidobasic properties, respectively, were synthesized for study of mechanism of uncoupling effect on oxidative phosphorylation. Their retention, influence on proton transport, abilities to SH--groups modify and to stimulate respiration in rat liver mitochondria, together with their physico-chemical properties, namely lipophilicity, acidobasicity and reactivity were characterized. The substitution of acidic hydrogen of the imino group resulted in the loss of both acidobasicity and uncoupling effect on oxidative phosphorylation. A decreased reactivity resulted from the substitutions of nitrile groups with the uncoupling activity remaining preserved.     

Physiological roles of the mitochondrial permeability transition pore

Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca²⁺ exchange/extrusion. The energy needs of these events are met in large part by mitochondrial production of ATP through the process of oxidative phosphorylation. The job of ATP production by the mitochondria is performed by the F₁F_O ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex. Although required for ATP production by mitochondria, recent findings have confirmed that the membrane-confined portion of the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane, uncoupling of oxidative phosphorylation and cell death. Recent advances in understanding the molecular components of mPTP and its regulatory mechanisms have determined that decreased uncoupling occurs in states of enhanced mitochondrial efficiency; relative closure of mPTP therefore contributes to cellular functions as diverse as cardiac development and synaptic efficacy.     

Fatty acid efficiency profile in uncoupling of <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> mitochondria

A profile of free fatty acid (FFA) specificity in Acanthamoeba castellanii mitochondrial uncoupling is described. The FFA uncoupling specificity was observed as different abilities to stimulate resting respiration, to decrease resting membrane potential, and to decrease oxidative phosphorylation efficiency. Tested unsaturated FFA (C18–20) were more effective as uncouplers and protonophores when compared to tested saturated FFA (C8–18), with palmitic acid (C16:0) as the most active. As FFA efficiency in mitochondrial uncoupling is related to physiological changes of fatty acid composition (and thereby FFA availability) during growth of amoeba cells, it could be a way to regulate the activity of an uncoupling protein and thereby the efficiency of oxidative phosphorylation during a cell life of this unicellular organism. Aleksandra Swida and Małgorzata Czarna contributed equally to this work.     

Crystal violet as an uncoupler of oxidative phosphorylation in rat liver mitochondria

Crystal violet exhibited characteristics of an uncoupler of oxidative phosphorylation, i.e. it released respiratory control, hindered ATP synthesis, enhanced ATPase activity, and produced swelling of isolated rat liver mitochondria. Maximal stimulation of respiration, ATPase activity, and swelling was observed at a concentration of 40 microM. The inhibition of State 3 respiration by oligomycin was released by crystal violet. High concentrations of crystal violet inhibited mitochondrial respiration. The uncoupling effect of crystal violet required inorganic phosphate and was abolished by N-ethylmaleimide. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of phosphate, the dye did not cause uncoupling, but its retention was much greater than in the presence of phosphate. Crystal violet is suggested to induce uncoupling by acting on the membrane, rather than by its electrophoretic transfer into the mitochondria.     

Alamethicin and synthetic peptide fragments as uncouplers of mitochondrial oxidative phosphorylation. Effect of chain length and change

Alamethicin, its derivatives and some synthetic fragments have been shown to be uncouplers of oxidative phosphorylation in rat liver mitochondria. A minimum peptide chain length of 13 residues is necessary for this activity. Peptide esters are more efficient uncouplers than the corresponding peptide acids. Esterification of the Glu(18) γ-COOH group in alamethicin does not diminish uncoupling activity. The structural requirements for uncoupling activity parallel those determined for ionophoretic action in small, unilamellar liposomes.     

Uncoupling activities of chalcones and dihydrochalcones on isolated mitochondria from potato tubers and mung bean hypocotyls

The uncoupling properties of 23 chalcones and dihydrochalcones were studied. Twelve compounds completely uncouple oxidative phosphorylation in mung bean hypocotyl and potato tuber mitochondria, four are weak uncouplers and seven are without effect. Usually, mung bean mitochondria are more sensitive to uncoupling agents than potato mitochondria. The uncoupling activity of chalcones and dihydrochalcones appears to be connected with the presence of hydrogen or hydroxyl groups in the 2′-position and hydrogen, hydroxyl or nitrate groups in the 4′-position. The α-β-unsaturated carbonyl system is not essential for activity. For the compounds which are not very lipophilic, substituents on the B-ring are without effect on the uncoupling properties. Phloretin appears to be an active uncoupler; its 6′-glucoside is without effect.     

In Phosphorylating <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> Mitochondria the Sensitivity of Uncoupling Protein Activity to GTP Depends on the Redox State of Quinone

In isolated Acanthamoeba castellanii mitochondria respiring in state 3 with external NADH or succinate, the linoleic acid-induced purine nucleotide-sensitive uncoupling protein activity is able to uncouple oxidative phosphorylation. The linoleic acid-induced uncoupling can be inhibited by a purine nucleotide (GTP) when quinone (Q) is sufficiently oxidized, indicating that in A. castellanii mitochondria respiring in state 3, the sensitivity of uncoupling protein activity to GTP depends on the redox state of the membranous Q. Namely, the inhibition of the linoleic acid-induced uncoupling by GTP is not observed in uninhibited state 3 respiration as well as in state 3 respiration progressively inhibited by complex III inhibitors, i.e., when the rate of quinol (QH₂)-oxidizing pathway is decreased. On the contrary, the progressive decrease of state 3 respiration by declining respiratory substrate availability (by succinate uptake limitation or by decreasing external NADH concentration), i.e., when the rate of Q-reducing pathways is decreased, progressively leads to a full inhibitory effect of GTP. Moreover, in A. castellanii mitochondria isolated from cold-treated cells, where a higher uncoupling protein activity is observed, the inhibition of the linoleic acid-induced proton leak by GTP is revealed for the same low values of the Q reduction level.     

Uncoupling of oxidative phosphorylation by fatty acids and detergents suppressed by ATP/ADP antiporter inhibitors

The effects of ATP/ADP-antiporter inhibitors on the uncoupling of oxidative phosphorylation by palmitic acid, detergents and protonophore FCCP in liver mitochondria were studied. The uncoupling activity of these compounds was estimated by their stimulating effect on succinate oxidation and H+ conductivity of the inner mitochondrial membrane in the presence of oligomycin. Carboxyatractylate and pyridoxal 5-phosphate suppressed the uncoupling effects of palmitic acid and anionic detergents but had no effect on the uncoupling action of the nonionic detergent Triton X-100, the cationic detergent CTAB and FCCP. The data obtained are discussed in terms of the putative role of the ATP/ADP-antiporter in the electrophoretic transport of hydrophobic anions from the mitochondria.     

Possible involvement of the 29 kDa protein in H+-ATPase in the action of cationic uncoupler of oxidative phosphorylation. Effect of the (o-phenanthroline)2-Cu2+ complex as a cationic uncoupler

The divalent cation (o-phenanthroline)2-Cu2+ complex was found to uncouple oxidative phosphorylation in mitochondria. Its uncoupling activity depended on inorganic phosphate (Pi) in the incubation medium, and was inhibited by the SH-reagent N-ethylmaleimide, and retarded by ATP. The uncoupling by the (o-phenanthroline)2-Cu2+ complex was suggested to be due to its modification of sulfhydryl groups in the 29 kDa protein in H+-ATPase.     

Uncoupling Effect of Fatty Acids in Halo- and Alkalotolerant Bacterium Bacillus pseudofirmus FTU

Natural uncouplers of oxidative phosphorylation, long-chain non-esterified fatty acids, cause uncoupling in the alkalo- and halotolerant bacterium Bacillus pseudofirmus FTU. The uncoupling effect in the bacterial cells was manifested as decrease of membrane potential and increase of respiratory activity. The membrane potential decrease was detected only in bacterial cells exhausted by their endogenous substrates. In proteoliposomes containing reconstituted bacterial cytochrome c oxidase, fatty acids caused a "mild" uncoupling effect by reducing membrane potential only at low rate of membrane potential generation. "Free respiration" induced by the "mild" uncouplers, the fatty acids, can be considered as possible mechanism responsible for adaptation of the bacteria to a constantly changed environment.