A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C₄R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C₁₂R1). Surprisingly, C₁₂R1 increased H⁺ conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C₄R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C₄R1. In experiments with HeLa cells, C₄R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C₄R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C₁₂R1. Thus, C₄R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.     

Effects of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> extract on heart and liver mitochondrial functions: mechanism(s) of action

Though extracts of Ginkgo biloba leaves (GBE) have a wide pharmacological application, little is known about GBE effects on mitochondria. In this work, effects of ethanolic GBE on the respiration of isolated rat heart and liver mitochondria were investigated. We found that GBE stimulates the pyruvate + malate-dependent State 2 respiration of heart mitochondria and decreases mitochondrial membrane potential. Uncoupling effect of GBE was found to be due to its protonophoric action and is likely to be mediated by the ATP/ADP-translocator and uncoupling proteins. The effect of GBE was less in liver than in heart mitochondria. State 3 respiration of heart mitochondria was slightly stimulated at low and depressed at higher GBE concentrations. Inhibition of State 3 respiration of heart mitochondria was not relieved by uncoupler indicating that GBE may inhibit the respiratory chain complexes or the substrate transport. However, Complex IV of the respiratory chain was not inhibited by GBE. H₂O₂ generation was attenuated by low concentration of GBE probably due to mild uncoupling. The data suggest that mild but not severe uncoupling activity of GBE may be important in providing pharmacological protection of cellular functions in pathological situations.     

Interaction of tetraphenylphosphonium and dodecyltriphenylphosphonium with lipid membranes and mitochondria

The permeability of a planar lipid membrane (composed of diphytanoylphosphatidylcholine) for tetraphenylphosphonium (TPP) was investigated. The observed level of the diffusion potential generated as a function of the TPP concentration gradient differed from the theoretically expected value, possibly due to proton leakage of the membrane mediated by the traces of fatty acids in the phospholipid forming the membrane. Using the molecular dynamics approach to study movement of TPP and dodecyltriphenylphosphonium (C₁₂TPP) with different affinity to the lipid bilayer through a bilayer lipid membrane, it was found that C₁₂TPP has a greater affinity to the membrane surface than TPP. However, the two cations have the same activation energy for transmembrane transfer. Interaction of TPP and C₁₂TPP with tightly-coupled mitochondria from the yeast Yarrowia lipolytica was also investigated. At low, micromolar concentrations, both cations are “relatively weak, mild uncouplers”, do not shunt electron transfer along the respiratory chain, do not disturb (damage) the inner mitochondrial membrane, and profoundly promote the uncoupling effect of fatty acids. At higher concentrations they inhibit respiration in state 3, and at much higher concentrations they induce swelling of mitochondria, possibly due to their detergent action.     

Mild uncoupling of respiration and phosphorylation as a mechanism providing nephro- and neuroprotective effects of penetrating cations of the SkQ family

It is generally accepted that mitochondrial production of reactive oxygen species is nonlinearly related to the value of the mitochondrial membrane potential with significant increment at values exceeding 150 mV. Due to this, high values of the membrane potential are highly dangerous, specifically under pathological conditions associated with oxidative stress. Mild uncoupling of oxidative phosphorylation is an approach to preventing hyperpolarization of the mitochondrial membrane. We confirmed data obtained earlier in our group that dodecylrhodamine 19 (C₁₂R1) (a penetrating cation from SkQ family not possessing a plastoquinone group) has uncoupling properties, this fact making it highly potent for use in prevention of pathologies associated with oxidative stress induced by mitochondrial hyperpolarization. Further experiments showed that C₁₂R1 provided nephroprotection under ischemia/reperfusion of the kidney as well as under rhabdomyolysis through diminishing of renal dysfunction manifested by elevated level of blood creatinine and urea. Similar nephroprotective properties were observed for low doses (275 nmol/kg) of the conventional uncoupler 2,4-dinitrophenol. Another penetrating cation that did not demonstrate protonophorous activity (SkQR4) had no effect on renal dysfunction. In experiments with induced ischemic stroke, C₁₂R1 did not have any effect on the area of ischemic damage, but it significantly lowered neurological deficit. We conclude that beneficial effects of penetrating cation derivatives of rhodamine 19 in renal pathologies and brain ischemia may be at least partially explained by uncoupling of oxidation and phosphorylation.     

Role of charge screening and delocalization for lipophilic cation permeability of model and mitochondrial membranes

The effects of the mitochondria-targeted lipophilic cation dodecyltriphenylphosphonium (C₁₂TPP, the charge is delocalized and screened by bulky hydrophobic residues) and those of lipophilic cations decyltriethylammonium bromide and cetyltrimethylammonium bromide (C₁₀TEA and C₁₆TMA, the charges are localized and screened by less bulky residues) on bilayer planar phospholipid membranes and tightly-coupled mitochondria from the yeast Yarrowia lipolytica have been compared. In planar membranes, C₁₂TPP was found to generate a diffusion potential as if it easily penetrates these membranes. In the presence of palmitate, C₁₂TPP induced H⁺ permeability like plastoquinonyl decyltriphenilphosphonium that facilitates transfer of fatty acid anions (Severin et al., PNAS, 2010, 107, 663–668). C₁₂TPP was shown to stimulate State 4 respiration of mitochondria and caused a mitochondrial membrane depolarization with a half-maximal effect at 6 μM. Besides, C₁₂TPP profoundly potentiated the uncoupling effect of endogenous or added fatty acids. C₁₀TEA and C₁₆TMA inhibited State 4 respiration and decreased the membrane potential, though at much higher concentrations than C₁₂TPP, and they did not promote the uncoupling action of fatty acids. These relationships were modeled by molecular dynamics. They can be explained by different membrane permeabilities for studied cations, which in turn are due to different availabilities of the positive charge in these cations to water dipoles.     

Electrogenic proton transport across lipid bilayer membranes mediated by cationic derivatives of rhodamine 19: comparison with anionic protonophores

Protonophores can be considered as candidates for anti-obesity drugs and tools to prevent excessive reactive oxygen species production in mitochondria by means of a limited decrease in the mitochondrial potential. Experimentally used protonophores are weak acids that can carry protons across a membrane in a neutral (protonated) form, and they come back in an anionic (deprotonated) form. A cationic derivative of rhodamine 19 and plastoquinone (SkQR1) was recently shown to possess uncoupling activity in mitochondria and in intact cells. In this article, we studied the mechanism of action of SkQR1 and its plastoquinone-lacking analog (C₁₂R1) on a planar bilayer lipid membrane by applying voltage jumps. The steady-state current was proportional to the C₁₂R1 concentration in a manner as if the monomeric form of the carrier were operative. As predicted by the carrier model, at high pH, when rhodamines were mainly deprotonated, the current changed immediately following a jump in the applied potential and then remained constant. By contrast, at low pH, the current relaxed from an initially high value to a lower value since the protonated carrier cations were redistributed in the membrane. An inverse pH dependence was revealed with the anionic protonophore CCCP. The dependence of the SkQR1 protonophorous activity on voltage exhibited an increase at high voltages, an effect that might facilitate mild (self-limited) uncoupling of mitochondria.     

Prevention of cardiolipin oxidation and fatty acid cycling as two antioxidant mechanisms of cationic derivatives of plastoquinone (SkQs)

The present state of the art in studies on the mechanisms of antioxidant activities of mitochondria-targeted cationic plastoquinone derivatives (SkQs) is reviewed. Our experiments showed that these compounds can operate as antioxidants in two quite different ways, i.e. (i) by preventing peroxidation of cardiolipin [Antonenko et al., Biochemistry (Moscow) 73 (2008) 1273–1287] and (ii) by fatty acid cycling resulting in mild uncoupling that inhibits the formation of reactive oxygen species (ROS) in mitochondrial State 4 [Severin et al. Proc. Natl. Acad. Sci. USA 107 (2009), 663–668]. The quinol and cationic moieties of SkQ are involved in cases (i) and (ii), respectively. In case (i) SkQH₂ interrupts propagation of chain reactions involved in peroxidation of unsaturated fatty acid residues in cardiolipin, the formed SkQ^? being reduced back to SkQH₂ by heme b_H of complex III in an antimycin-sensitive way. Molecular dynamics simulation showed that there are two stable conformations of SkQ1 with the quinol residue localized near peroxyl radicals at C₉ or C₁₃ of the linoleate residue in cardiolipin. In mechanism (ii), fatty acid cycling mediated by the cationic SkQ moiety is involved. It consists of (a) transmembrane movement of the fatty acid anion/SkQ cation pair and (b) back flows of free SkQ cation and protonated fatty acid. The cycling results in a protonophorous effect that was demonstrated in planar phospholipid membranes and liposomes. In mitochondria, the cycling gives rise to mild uncoupling, thereby decreasing membrane potential and ROS generation coupled to reverse electron transport in the respiratory chain. In yeast cells, dodecyltriphenylphosphonium (С₁₂TPP), the cationic part of SkQ1, induces uncoupling that is mitochondria-targeted since С₁₂TPP is specifically accumulated in mitochondria and increases the H⁺ conductance of their inner membrane. The conductance of the outer cell membrane is not affected by С₁₂TPP.     

Efficiency of mitochondrial uncoupling by modified butyltriphenylphosphonium cations and fatty acids correlates with lipophilicity of cations: Protonophoric vs leakage mechanisms

In order to determine the share of protonophoric activity in the uncoupling action of lipophilic cations a number of analogues of butyltriphenylphosphonium with substitutions in phenyl rings (C₄TPP-X) were studied on isolated rat liver mitochondria and model lipid membranes. An increase in the rate of respiration and a decrease in the membrane potential of isolated mitochondria were observed for all the studied cations, the efficiency of these processes was significantly enhanced in the presence of fatty acids and correlated with the octanol-water partition coefficient of the cations. The ability of C₄TPP-X cations to induce proton transport across the lipid membrane of liposomes loaded with a pH-sensitive fluorescent dye increased also with their lipophilicity and depended on the presence of palmitic acid in the liposome membrane. Of all the cations, only butyl[tri(3,5-dimethylphenyl)]phosphonium (C₄TPP-diMe) was able to induce proton transport by the mechanism of formation of a cation-fatty acid ion pair on planar bilayer lipid membranes and liposomes. The rate of oxygen consumption by mitochondria in the presence of C₄TPP-diMe increased to the maximum values corresponding to conventional uncouplers; for all other cations the maximum uncoupling rates were significantly lower. We assume that the studied cations of the C₄TPP-X series, with the exception of C₄TPP-diMe at low concentrations, cause nonspecific leak of ions through lipid model and biological membranes which is significantly enhanced in the presence of fatty acids.     

Rat liver mitochondria impairments under acute carbon tetrachloride-induced intoxication. Effects of melatonin

The aim of the present work was to investigate the mechanisms of oxidative damage of rat liver mitochondria in vitro, under hypochlorous acid (HOCl)-induced oxidative stress, and in vivo, under acute carbon tetrachloride-induced intoxication in rats. Hypochlorous acid (50–300 μM), the main inflammatory agent, inhibited liver mitochondria respiratory activity and caused uncoupling in the respiratory and phos-porylation processes. The toxic damage of rat liver after 24 h of acute carbon tetrachloride-induced intoxication (4 g/kg, intragastrically) was accompanied by a significant reduction in succinate- and glutamate-dependent respiration rate in state 3 (by 65%, p < 0.001, and by 50%, p < 0.01, respectively). The respiration control ratio approached 1, reflecting the loss of respiration control. The phosphorylation coefficient significantly decreased due to uncoupling of the oxidation and phosphorylation processes. The mitochondrial alterations were associated with oxidation of intramitochondrial GSH by 25% (p < 0.05), the marked inhibition of succinate dehydrogenase (complex II) by 35% (p < 0.05), and the rise of blood plasma nitric oxide level by 45% (p < 0.05). The impairment of mitochondrial respiratory function may result from the inhibition of enzymatic activities in the respiratory chain and the damage of mitochondrial membrane during intoxication and plays a key role in the development of the CCl₄-induced hepatotoxicity. Melatonin administration under CCl₄-induced intoxication (three times at a dose of 10 mg/kg) increased the rate of succinate oxidation in state 3 by 30% (p < 0.05) and reversed the increase in glutathione peroxidase activity. Melatonin prevented an elevation of nitric oxide level in the blood plasma of intoxicated animals but did not protect mitochondrial functions under acute intoxication.     

Interaction of yeast mitochondria with fatty acids and mitochondria-targeted lipophilic cations

The effect of fatty acids and mitochondria-targeted lipophilic cations (SkQ1, SkQ3, MitoQ, and C₁₂TPP) on tightly-coupled mitochondria from yeasts Dipodascus (Endomyces) magnusii and Yarrowia lipolytica was investigated. Micromolar concentrations of saturated and unsaturated fatty acids were found to decrease the membrane potential, which was recovered almost totally by ATP and BSA. At low, micromolar concentrations, mitochondria-targeted lipophilic cations are “relatively weak, mild uncouplers”, at higher concentrations they inhibit respiration in state 3, and at much higher concentrations they induce swelling of mitochondria, possibly due to their prooxidant and detergent action. At very low, not uncoupling concentrations, mitochondria-targeted lipophilic cations profoundly promote (potentiate) the uncoupling effect of fatty acids. It is conceivable that the observed uncoupling effect of lipophilic cations can be, at least partially, due to their interactions with the endogenous pool of fatty acids.     

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.     

The interaction of flavonoids with mitochondria: effects on energetic processes

The study addressed aspects of energetics of isolated rat liver mitochondria exposed to the flavonoids quercetin, taxifolin, catechin and galangin, taking into account influences of the 2,3 double bond/3-OH group and 4-oxo function on the C-ring, and o-di-OH on the B-ring of their structures, as well as mitochondrial mechanisms potentially involved in cell necrosis and apoptosis. The major findings/hypothesis, were: The 2,3 double bond/3-OH group in conjugation with the 4-oxo function on the C-ring in the flavonoid structure seems favour the interaction of these compounds with the mitochondrial membrane, decreasing its fluidity either inhibiting the respiratory chain of mitochondria or causing uncoupling; while the o-di-OH on the B-ring seems favour the respiratory chain inhibition, the absence of this structure seems favour the uncoupling activity. The flavonoids not affecting the respiration of mitochondria, induced MPT. The ability of flavonoids to induce the release of mitochondria-accumulated Ca(2+) correlated well with their ability to affect mitochondrial respiration on the one hand, and their inability to induce MPT, on the other. The flavonoids causing substantial respiratory chain inhibition or mitochondrial uncoupling, quercetin and galangin, respectively, also decreased the mitochondrial ATP levels, thus suggesting an apparent higher potential for necrosis induction in relation to the flavonoids inducing MPT, taxifolin and cathechin, which did not decrease significantly the ATP levels, rather suggesting an apparent higher potential for apoptosis induction.     

Influence of CSP 310 and CSP 310-like proteins from cereals on mitochondrial energetic activity and lipid peroxidation in vitro and in vivo

Background  

The development of chilling and freezing injury symptoms in plants is known to frequently coincide with peroxidation of free fatty acids. Mitochondria are one of the major sources of reactive oxygen species during cold stress. Recently it has been suggested that uncoupling of oxidation and phosphorylation in mitochondria during oxidative stress can decrease ROS formation by mitochondrial respiratory chain generation. At the same time, it is known that plant uncoupling mitochondrial protein (PUMP) and other UCP-like proteins are not the only uncoupling system in plant mitochondria. All plants have cyanide-resistant oxidase (AOX) whose activation causes an uncoupling of respiration and oxidative phosphorylation. Recently it has been found that in cereals, cold stress protein CSP 310 exists, and that this causes uncoupling of oxidation and phosphorylation in mitochondria.     

Effect of salicylic acid on the metabolic activity of plant mitochondria

The effects of salicylic acid (SA) on mitochondrial respiration and generation of membrane potential across the inner membrane of mitochondria isolated from stored taproots of sugar beet (Beta vulgaris L.) and etiolated seedling cotyledons of yellow lupine (Lupinus luteus L.) were studied. When malate was oxidized in the presence of glutamate, low SA concentrations (lower than 1.0 mM) exerted predominantly uncoupling action on the respiration of taproot mitochondria: they activated the rate of oxygen uptake in State 4 (in the absence of ADP) and did not affect oxidation in State 3 (in the presence of ADP). In contrast, in lupine cotyledon mitochondria these SA concentrations inhibited oxygen uptake in the presence of ADP and much weaker activated substrate oxidation in State 4. Thus, SA (0.5 mM) reduced the respiratory control ratio according to Chance (RCR) by 25% in the taproots and 35% in cotyledons. When the concentration of phytohormone was increased (above 1.0 mM), malate oxidation in State 3 was inhibited and in State 4 — activated independently of the plant material used. In this case, the values of RCR and ADP/O were reduced by 50–60%. The effect of high SA concentrations (2 mM and higher) on malate oxidation depended on the duration of incubation and had a biphasic pattern: the initial activation of oxygen uptake was later replaced by its inhibition. The parallel studying the SA effect on the generation of membrane potential (ΔΨ) at malate oxidation in the mitochondria of beet taproots and lupine cotyledons showed that ΔΨ dissipation was observed because of SA uncoupling and inhibiting action on respiration. The degree of ΔΨ dissipation depended on the phytohormone concentration and duration on mitochondria treatment, especially at its high concentrations. In general, a correlation was found between the effects of SA on mitochondrial respiration and ΔΨ values in the coupling membranes. Furthermore, these results show that the responses of mitochondria to SA were determined not only by its concentration but also by treatment duration and evidently by the sensitivity to the phytohormone of mitochondria isolated from different plant tissues.     

Decrease in mitochondrial energy coupling by thyroid hormones: a physiological effect rather than a pathological hyperthyroidism consequence

The effect of the in vivo thyroid status on mitochondrial membrane potential (ΔΨ_m) in isolated rat hepatocytes was studies by means of a cytofluorimetric technique and the ΔΨ_m-specific probe JC-1. It is shown that the ΔΨ_m level decreases in the order hypothyroid>euthyroid>hyperthyroid. Polarographic measurement of the hepatocyte respiratory rates revealed an opposite trend of values: the highest respiratory rate in hepatocytes from hyperthyroid animals, the lowest in those from hypothyroid ones. This means that mitochondrial energy coupling is highest in hypothyroid hepatocytes and lowest in hyperthyroid hepatocytes. 6-Ketocholestanol added to hepatocytes failed to counterbalance the uncoupling effect of thyroid hormones on ΔΨ_m and respiration rate. Under the same conditions, 6-ketocholestanol appeared to be effective in recoupling of respiration uncoupled by low concentrations of the artificial protonophore FCCP. The mechanism and possible physiological functions of the thyroid hormone-induced decrease in mitochondrial energy coupling are discussed.     

Respiratory uncoupling by UCP1 and UCP2 and superoxide generation in endothelial cell mitochondria

Mitochondria represent a major source of reactive oxygen species (ROS), particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins (UCPs) is to decrease mitochondrial membrane potential and thereby protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by UCPs. Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance spectroscopy, was approximately fourfold greater during state 4 compared with state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria, as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, despite uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-(trifluoromethoxyl)-phenyl-hydrazone. In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria but that uncoupling by UCP1 or UCP2 does not prevent acute substrate-driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.     

Derivatives of rhodamine 19 as mild mitochondria-targeted cationic uncouplers

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.     

In VivoInterference of Paromomycin with Mitochondrial Activity ofLeishmania

Paromomycin is an aminocyclitol aminoglycoside antibiotic used for the treatment of leishmaniasis. In view of the central role of mitochondria in cellular energetics and metabolism, its effect onin vivomitochondrial activities ofLeishmania donovanipromastigotes—the parasite flagellate form—was investigated. The approach used flow cytometry, amperometric measure of O₂consumption, and, as a global estimate of mitochondrial dehydrogenases, thiazolyl blue reduction (MTT test); somein vitrocontrols were also made. When added to promastigote cultures for 24–72 h at 150–200 μM(= LC₅₀), paromomycin doubled the generation time, inhibited respiration, and lowered its associated electric potential difference across mitochondrial membranes, as measured by rhodamine 123 fluorescence. The chemical analogue neomycin was ineffective. Furthermore, thein vivomitochondrial dehydrogenase activities were lower, seemingly because of the shortage of respiratory substrates. Indeed, succinate addition to paromomycin-treated cultures partly restored mitochondrial membrane potential. However, no immediate effect of paromomycin on respiration was observed, neither inhibition of redox chain nor increase of membrane permeability (uncoupling). It is proposed that paromomycin acts at a metabolic level upstream of the respiratory chain itself. This would have the observed delayed consequence because the cell energy supply would progressively decline since it depends upon the proton gradient—viz., membrane potential—generated by respiration. In conclusion, paromomycin is an antibiotic affecting the cell's energetic metabolism; the respiratory dysfunction it induces may be a crucial aspect of its action againstLeishmaniaand possibly other cells.     

A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species

Carbon monoxide (CO), produced during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator in mammalian cells. Here we show that precise delivery of CO to isolated heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) uncouples respiration. Addition of low-micromolar concentrations of CORM-3 (1–20 μM), but not an inactive compound that does not release CO, significantly increased mitochondrial oxygen consumption rate (State 2 respiration) in a concentration-dependent manner. In contrast, higher concentrations of CORM-3 (100 μM) suppressed ADP-dependent respiration through inhibition of cytochrome c oxidase. The uncoupling effect mediated by CORM-3 was inhibited in the presence of the CO scavenger myoglobin. Moreover, this effect was associated with a gradual decrease in membrane potential (ψ) over time and was partially reversed by malonate, an inhibitor of complex II activity. Similarly, inhibition of uncoupling proteins or blockade of adenine nucleotide transporter attenuated the effect of CORM-3 on both State 2 respiration and Δψ. Hydrogen peroxide (H₂O₂) produced by mitochondria respiring from complex I-linked substrates (pyruvate/malate) was increased by CORM-3. However, respiration initiated via complex II using succinate resulted in a fivefold increase in H₂O₂ production and this effect was significantly inhibited by CORM-3. These findings disclose a counterintuitive action of CORM-3 suggesting that CO at low levels acts as an important regulator of mitochondrial respiration.     

Acute administration of 3,5-diiodo-L-thyronine to hypothyroid rats stimulates bioenergetic parameters in liver mitochondria

The role of 3,5-diiodo-L-thyronine (T₂), initially considered only a 3,3′,5-triiodo-L-thyronine (T₃) catabolite, in the bioenergetic metabolism is of growing interest. In this study we investigated the acute effects (within 1 h) of T₂ administration to hypothyroid rats on liver mitochondria fatty acid uptake and β-oxidation rate, mitochondrial efficiency (by measuring proton leak) and mitochondrial oxidative damage (by determining H₂O₂ release). Fatty acid uptake into mitochondria was measured assaying carnitine palmitoyl transferase (CPT) I and II activities, and fatty acid β-oxidation using palmitoyl-CoA as a respiratory substrate. Mitochondrial fatty acid pattern was defined by gas-liquid chromatography. In hypothyroid + T₂ vs hypothyroid rats we observed a raise in the serum level of nonesterified fatty acids (NEFA), in the mitochondrial CPT system activity and in the fatty acid β-oxidation rate. A parallel increase in the respiratory chain activity, mainly from succinate, occurs. When fatty acids are chelated by bovine serum albumin, a T₂-induced increase in both state 3 and state 4 respiration is observed, while, when fatty acids are present, mitochondrial uncoupling occurs together with increased proton leak, responsible for mitochondrial thermogenesis. T₂ administration decreases mitochondrial oxidative stress as determined by lower H₂O₂ production. We conclude that in rat liver mitochondria T₂ acutely enhances the rate of fatty acid β-oxidation, and the activity of the downstream respiratory chain. The T₂-induced increase in proton leak may contribute to mitochondrial thermogenesis and to the reduction of oxidative stress. Our results strengthen the previously reported ability of T₂ to reduce adiposity, dyslipidemia and to prevent liver steatosis.