Transmembrane potential of liver mitochondria from hexachlorobenzene- and iron-treated rats

The respiratory parameters and the membrane of liver mitochondria from rats treated with either hexachlorobenzene, iron or hexachlorobenzene plus iron, to induce experimental porphyria, have been studied. Partial uncoupling of oxidative phosphorylation has been observed in mitochondria from hexachlorobenzen- and hexachlorobenzene plus iron-treated rats. Direct evidence has been pressented that this uncoupling is due to the action of pentochlorophenol endogenously formed by metabolism of hexachlorobenzene. No irreversible damage of mitochondrial membrane has been revealed under both these conditions. Normal oxidative phosphorylation has bee found in mitochondria from rats treated with iron alone. In contrast, they presented an anomalous membrane potential, fully restored by oligomycin. A possible involvement of lipid peroxidation process, induced by iron, in causing these abnormalities has been suggested.     

Structural and functional properties of rat liver mitochondria in hexachlorobenzene induced experimental porphyria

A possible link between changes in iron and porphyrin content in liver mitochondria, from rats treated with either hexachlorobenzene, iron, or hexachlorobenzene plus iron, as a function of treatment time and their structural-functional properties, has been investigated. Normal oxidative phosphorylation in mitochondria from rats treated with iron has been shown. By contrast a significant and constant uncoupling of the phosphorylative process, fully reversed by albumin, in mitochondria from rats treated with hexachlorobenzene and hexachlorobenzene plus iron has been presented. A possible involvement of pentachlorophenol in causing these abnormalities has been proposed.     

The effect of iron overload on the mitochondrial porphyrin level in the hexachlorobenzene induced experimental porphyria

Liver mitochondria isolated from rats treated with hexachlorobenzene plus iron, present a lower content of total porphyrin in respect to that of mitochondria from rats fed hexachlorobenzene alone. The in vitro mitochondrial porphyrin accumulation processes have been studied in mitochondria from iron loaded rats. It has been found that under these conditions the active porphyrin uptake process, which is driven by the K+ transmembrane gradient, is maximally inhibited in the presence of pentachlorophenol at a concentration similar to that found in vivo in the hexachlorobenzene experimental porphyria. By contrast the same degree of inhibition is presented by control mitochondria only in the presence of pentachlorophenol plus valinomycin, a condition which collapses the transmembrane K+ gradient. A strict correlation between porphyrin uptake and K+ concentration has been found in control as well as in iron treated mitochondria. A possible involvement of peroxidative reactions in the mitochondrial membranes has been proposed as a cause of the changes in the permeability properties of the mitochondrial membranes in the experimental chronic hepatic porphyria under conditions of iron overload.     

Effects of hexachlorobenzene and iron loading on rat liver mitochondria

The effects of hexachlorobenzene treatment and simultaneous iron-overload on the iron and porphyrin content of rat liver and rat liver mitochondria have been examined. In order to assess damages to the mitochondrial membrane occuring with these treatments, the content of malondialdehyde and selected functional properties of mitochondria were compared with those from control animals. Prolonged intake of hexachlorobenzene (8 weeks) resulted in a striking increased level of porphyrins together with a moderate increase in iron concentration. Simultaneous administration of hexachlorobenzene and iron-dextran caused the porphyrin level to reach 25% of the amount induced by hexachlorobenzene alone. The iron concentrations in liver as well as in liver mitochondria are also decreased under these conditions, as compared to the effect of iron-dextran. In contrast, the effects of hexachlorobenzene combined with iron-dextran on mitochondrial oxidative phosphorylation and malondialdehyde content are greater than those of either hexachlorobenzene or iron-dextran. These data suggest that porphyrin accumulation per se causes little deleterious effect and that both agents administered together act synergistically in causing damage to the mitochondrial membrane.     

Uncoupling proteins in mitochondria of plants and some microorganisms.

Uncoupling proteins, members of the mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. Since 1995, it has been shown that the uncoupling protein is present in many higher plants and some microorganisms like non-photosynthetic amoeboid protozoon, Acanthamoeba castellanii and non-fermentative yeast Candida parapsilosis. In mitochondria of these organisms, uncoupling protein activity is revealed not only by stimulation of state 4 respiration by free fatty acids accompanied by decrease in membrane potential (these effects being partially released by ATP and GTP) but mainly by lowering ADP/O ratio during state 3 respiration. Plant and microorganism uncoupling proteins are able to divert very efficiently energy from oxidative phosphorylation, competing for deltamicroH+ with ATP synthase. Functional connection and physiological role of uncoupling protein and alternative oxidase, two main energy-dissipating systems in plant-type mitochondria, are discussed.     

Acute knockdown of uncoupling protein-2 increases uncoupling via the adenine nucleotide transporter and decreases oxidative stress in diabetic kidneys

Increased O(2) metabolism resulting in chronic hypoxia is common in models of endstage renal disease. Mitochondrial uncoupling increases O(2) consumption but the ensuing reduction in mitochondrial membrane potential may limit excessive oxidative stress. The present study addressed the hypothesis that mitochondrial uncoupling regulates mitochondria function and oxidative stress in the diabetic kidney. Isolated mitochondria from kidney cortex of control and streptozotocin-induced diabetic rats were studied before and after siRNA knockdown of uncoupling protein-2 (UCP-2). Diabetes resulted in increased UCP-2 protein expression and UCP-2-mediated uncoupling, but normal mitochondria membrane potential. This uncoupling was inhibited by GDP, which also increased the membrane potential. siRNA reduced UCP-2 protein expression in controls and diabetics (-30-50%), but paradoxically further increased uncoupling and markedly reduced the membrane potential. This siRNA mediated uncoupling was unaffected by GDP but was blocked by ADP and carboxyatractylate (CAT). Mitochondria membrane potential after UCP-2 siRNA was unaffected by GDP but increased by CAT. This demonstrated that further increased mitochondria uncoupling after siRNA towards UCP-2 is mediated through the adenine nucleotide transporter (ANT). The increased oxidative stress in the diabetic kidney, manifested as increased thiobarbituric acids, was reduced by knocking down UCP-2 whereas whole-body oxidative stress, manifested as increased circulating malondialdehyde, remained unaffected. All parameters investigated were unaffected by scrambled siRNA. In conclusion, mitochondrial uncoupling via UCP-2 regulates mitochondria membrane potential in diabetes. However, blockade of the diabetes-induced upregulation of UCP- 2 results in excessive uncoupling and reduced oxidative stress in the kidney via activation of ANT.     

Cold-induced changes in the energy coupling and the UCP3 level in rodent skeletal muscles

The mechanism of thermoregulatory uncoupling of respiration and phosphorylation in skeletal muscles has been studied. It is found that 24 h cold exposure results in (i) a 3-fold increase in the amount of UCP3 protein in rat skeletal muscle mitochondria, and (ii) pronounced lowering of the membrane potential in isolated rat or mouse skeletal muscle mitochondria. The decrease in membrane potential is reversed by adding bovine serum albumin. Cold exposure is also found to sensitize the membrane potential to the uncoupling action of added fatty acid (laurate). After laurate addition, the recoupling effects of GDP and carboxyatractylate decrease whereas that of albumin increases in mitochondria from cold-treated rats or mice. Changes similar to those induced by cold can be initiated by the in vivo addition of thyroxine. Cold exposure does not affect energy coupling in liver mitochondria. The possible involvement of UCP3 isoforms in nucleotide-sensitive and -insensitive uncoupling is discussed.     

Regulation of thyroid hormones of the interaction of mitochondria with low-molecular weight cytoplasmic mediators, induced by phosphate- dependent transport of K+ and H+ ions through the mitochondrial inner membrane

In vivo thyroid hormones control the binding to mitochondria of low molecular weight water-soluble cytoplasmic mediators that are capable to induce oxidative phosphorylation uncoupling, by increasing the sensitivity of mitochondria to the effects of these mediators. In hyperthyroid rat liver mitochondria cytoplasmic mediators stimulate the phosphate-dependent transport of K+ and H+ in a greater degree than in liver mitochondria of control rats. The increase in the oxidative phosphorylation uncoupling by cytoplasmic mediators is one of mechanisms of thermogenesis stimulation by thyroid hormones.     

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.     

Toxic action of 2'-chloro-2,4-dinitro-5',6-di(trifluoromethyl) diphenylamine in the rat.

2'-Chloro-2,4-dinitro-5',6-di(trifluoromethyl)diphenylamine (CDTD) is a potent uncoupler of oxidative phosphorylation in isolated rat liver or brain mitochondria. The concentration of CDTD causing 50% uncoupling in vitro is dependent on the mitochdonrial protein concentration and is 2 nM at 0.9 mg protein/ml for rat liver mitochondria. Oxidative phosphorylation can be restored to CDTD uncoupled liver mitochondria by the addition of a 10 000-fold molar excess of bovine serum albumin to DCTD. Rats given a lethal dose (7.0 mumol/kg) of CDTD intrapertioneally show signs of toxicity typical of uncoupling agents. Mitochondria isolated from the livers of these rats show almost complete inhibition of ATP synthesis and mitochondria obtained from the livers of rats at various times after a single oral dose show maximal inhibition of ATP synthesis 4 h after dosing with complete recovery by about 24 h. A single oral administration of 58 mumol/kg or above, but not intraperitoneal injection, of CDTD into rats produced an increase in the water content of the brain and spinal cord. The additional fluid has been shown to contain Na+ ions. The increase in cerebral fluid is dose related, no effect being seen at 23 mumol/kg. This extra fluid is thought to be responsible for the hind limb weakness observed in these rats. These observations suggest that there are two facets to CDTD toxicity: early deaths (within 2 h), which appear to be due to uncoupling of oxidative phosphorylation, and delayed deaths, 2--3 days after dosing which are probably related to an increase in fluid in the brain and spinal cord.     

Carboxyatractylate-sensitive uncoupling in liver mitochondria from ground squirrels during hibernation and arousal

Energy coupling parameters of liver mitochondria from hibernating and arousing ground squirrels have been studied. In the oligomycin-treated mitochondria, carboxyatractylate, an inhibitor of the ATP/ADP-antiporter, is shown to decrease the respiration rate, to increase the membrane potential and to lower the rate of the membrane-potential discharge after the addition of cyanide to liver mitochondria from hibernating and arousing animals. BSA effectively substitutes for carboxyactactylate so that carboxyactactylate, added after BSA, has no effect. In mitochondria from hibernating animals, the maximal respiration rate in the presence of DNP and the rate of the membrane potential discharge in its absence are much lower than in those from arousing animals. It has been concluded that upon arousal of the animals from hibernation, the uncoupling of oxidative phosphorylation, mediated by free fatty acids and ATP/ADP-antiporter, parallels the respiratory chain activation.     

Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin E-deficient rats

Few, if any, studies have examined the effect of vitamin E deficiency on brain mitochondrial oxidative phosphorylation. The latter was studied using brain mitochondria isolated from control and vitamin E-deficient rats (13 months of deficiency) after exposure to iron, an inducer of oxidative stress. Mitochondria were treated with iron (2 to 50 microM) added as ferrous ammonium sulfate. Rates of state 3 and state 4 respiration, respiratory control ratios, and ADP/O ratios were not affected by vitamin E deficiency alone. However, iron uncoupled oxidative phosphorylation in vitamin E-deficient mitochondria, but not in controls. In vitamin E-deficient mitochondria, iron decreased ADP/O ratios and markedly stimulated state 4 respiration; iron had only a modest effect on these parameters in control mitochondria. Thus, vitamin E may have an important role in sustaining oxidative phosphorylation. Low concentrations of iron (2 to 5 microM) oxidized mitochondrial tocopherol that exists in two pools. The release of iron in brain may impair oxidative phosphorylation, which would be exacerbated by vitamin E deficiency. The results are important for understanding the pathogenesis of human brain disorders known to be associated with abnormalities in mitochondrial function as well as iron homeostasis (e.g., Parkinson's disease).     

Mitochondrial uncoupling proteins: new insights from functional and proteomic studies

Mitochondria are the major sites of ATP synthesis through oxidative phosphorylation, a process that is weakened by proton leak. Uncoupling proteins are mitochondrial membrane proteins specialized in inducible proton conductance. They dissipate the proton electrochemical gradient established by the respiratory chain at the expense of reducing substrates. Several physiological roles have been suggested for uncoupling proteins, including roles in the control of the cellular energy balance and in preventive action against oxidative stress. This review focuses on new leads emerging from comparative proteomics about the involvement of uncoupling protein in the mitochondrial physiology. A brief overview on uncoupling proteins and on proteomics applied to mitochondria is also presented herein.     

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.     

Regulation of oxidative phosphorylation, K+ ions transport and the volume of mitochondrial matrix by cytoplasmic glycopeptide and Ca2+ ions

A thermostable low molecular weight glycopeptide containing syalic acids, which uncouples mitochondrial oxidative phosphorylation, has been detected, isolated and purified from rat liver cytoplasm. In the presence of the glycopeptide, oxidative phosphorylation in rat liver mitochondria is uncoupled by low physiological concentrations of Ca2+, which otherwise do not have any appreciable effect on the mitochondria. Oxidative phosphorylation uncoupling by the glycopeptide is accompanied by an increase of the mitochondrial volume. This process has a limited amplitude and is regulated by changes in Ca2+ concentration in the extramitochondrial space. The glycopeptide has been shown to induce K+ transport across the inner mitochondrial membrane, this effect is enhanced by Ca2+.     

Oxidative phosphorylation, Ca(2+) transport, and fatty acid-induced uncoupling in malaria parasites mitochondria

Respiration, oxidative phosphorylation, calcium uptake, and the mitochondrial membrane potential of trophozoites of the malaria parasite Plasmodium berghei were assayed in situ after permeabilization with digitonin. ADP promoted an oligomycin-sensitive transition from resting to phosphorylating respiration. Respiration was sensitive to antimycin A and cyanide. The capacity of trophozoites to sustain oxidative phosphorylation was additionally supported by the detection of an oligomycin-sensitive decrease in mitochondrial membrane potential induced by ADP. Phosphorylation of ADP could be obtained in permeabilized trophozoites in the presence of succinate, citrate, alpha-ketoglutarate, glutamate, malate, dihydroorotate, alpha-glycerophosphate, and N,N,N',N'-tetramethyl-p-phenylenediamine. Ca(2+) uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca(2+) transport system in these mitochondria. An uncoupling effect of fatty acids was partly reversed by bovine serum albumin, ATP, or GTP and not affected by atractyloside, ADP, glutamate, or malonate. Evidence for the presence of a mitochondrial uncoupling protein in P. berghei was also obtained by using antibodies raised against plant uncoupling mitochondrial protein. Together these results provide the first direct biochemical evidence of mitochondrial function in ATP synthesis and Ca(2+) transport in a malaria parasite and suggest the presence of an H(+) conductance in trophozoites similar to that produced by a mitochondrial uncoupling protein.     

The bulk of UCP3 expressed in yeast cells is incompetent for a nucleotide regulated H+ transport

The impact of uncoupling protein (UCP) 1, UCP3 and UCP3s expressed in yeast on oxidative phosphorylation, membrane potential and H⁺ transport is determined. Intracellular ATP synthesis is inhibited by UCP3, much more than by UCP1, while similar levels of UCP3 and UCP1 exist in the mitochondrial fractions. Measurements of membrane potential and H⁺ efflux in isolated mitochondria show that, different from UCP1, with UCP3 and UCP3s there is a priori a preponderant uncoupling not inhibited by GDP. The results are interpreted to show that UCP3 and UCP3s in yeast mitochondria are in a deranged state causing uncontrolled uncoupling, which does not represent their physiological function.     

Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential

Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of ROS (reactive oxygen species). This proton leak can be induced by the lipid peroxidation products of ROS, such as HNE (4-hydroxynonenal). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and ANT (adenine nucleotide translocase), thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We have found that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent on incubation with respiratory substrate. In the presence of HNE, mitochondria energized with succinate became progressively more leaky to protons over time compared with mitochondria in the absence of either HNE or succinate. Energized mitochondria must attain a high membrane potential to allow HNE to activate uncoupling: a drop of 10-20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), ANT (64%) and other pathways (25%). Our findings have shown that exogenous HNE only activates uncoupling at high membrane potential. These results suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production.     

Functional characterization of the mitochondrial uncoupling proteins from the white shrimp Litopenaeus vannamei

Mitochondrial uncoupling proteins (UCPs) play an essential role in dissipating the proton gradient and controlling the mitochondrial inner membrane potential. When active, UCPs promote proton leak across the inner membrane, oxidative phosphorylation uncoupling, oxygen uptake increase and decrease the ATP synthesis. Invertebrates possess only isoforms UCP4 and UCP5, however, the role of these proteins is not clear in most species since it may depend on the physiological needs of each animal. This study presents the first functional characterization of crustacean uncoupling proteins from the white shrimp Litopenaeus vannamei LvUCP4 and LvUCP5. Free radicals production in various shrimp organs/tissues was first evaluated, and mitochondria were isolated from shrimp pleopods. The oxygen consumption rate, membrane potential and proton transport of the isolated non-phosphorylating mitochondria were used to determine LvUCPs activation/inhibition. Results indicate that UCPs activity is stimulated in the presence of 4-hydroxyl-2-nonenal (HNE) and myristic acid, and inhibited by the purine nucleotide GDP. A hypoxia/re-oxygenation assay was conducted to determine whether UCPs participate in shrimp mitochondria response to oxidative stress. Isolated mitochondria from shrimp at re-oxygenation produced large quantities of hydrogen peroxide and higher levels of both LvUCPs were immunodetected. Results suggest that, besides the active response of the shrimp antioxidant system, UCP-like activity is activated after hypoxia exposure and during re-oxygenation. LvUCPs may represent a mild uncoupling mechanism, which may be activated before the antioxidant system of cells, to early control reactive oxygen species production and oxidative damage in shrimp.