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.     

Duramycin effects on the structure and function of heart mitochondria. II. Energy conversion reactions.

The polypeptide antibiotic duramycin has been reported to interact selectively with phosphatidylethanolamine (PE) and monogalactosyldiacylglycerol (Navarro et al., 1985, Biochemistry 24, 4645-4650). PE is a major component of mitochondrial membranes. Duramycin was used to probe the role of PE in mitochondrial energy conversion reactions with the following results: (i) Duramycin uncoupled mitochondrial respiration, decreasing the respiratory control ratio to 1 at 5 microM. At concentrations of duramycin in excess of 10 microM, ADP addition inhibited electron transport. (ii) Duramycin inhibited oxidative phosphorylation (C50 less than 2 microM). (iii) Duramycin stimulated mitochondrial ATP hydrolysis modestly. The antibiotic was 7- to 16-fold less effective in this regard than concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone (F-CCP) which produced comparable uncoupling. (iv) Duramycin inhibited uncoupled ATPase activity (C50 = 8 microM). Inhibition of the ATPase activity of intact mitochondria was blocked by 1 mM MgCl2 and 5 mM CaCl2; inhibition persisted in sub-mitochondrial particles assayed in the presence of 3 mM MgCl2. The effects on mitochondrial function of free fatty acids (FFA) and duramycin are similar in many respects. It is suggested that duramycin, like FFA, uncouples via a nonclassical mechanism, possibly by disrupting intramembrane H+ transfer between redox and ATPase complexes. In addition, interaction of duramycin, either direct or indirect, with the F0 moiety of the mitochondrial ATPase and with one or more components of the respiratory electron transport chain is proposed.     

The stimulation of hepatic oxidative phosphorylation following dexamethasone treatment of rats

The effect of short-term treatment of rats with the synthetic glucocorticoid, dexamethasone, on mitochondrial oxidative phosphorylation has been examined. Treatment of rats for 3 h increased the oxidative capacity of the subsequently isolated mitochondria such that they displayed increased uncoupled and State 3 rates of respiration with NAD-linked substrates, succinate or durohydroquinone. The oxidation of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine was unaffected. No change was apparent in the activity of a variety of dehydrogenase enzymes nor was there any increase in the mitochondrial content of cytochromes a, b, c1 or c. The uncoupler-dependent ATPase activity of the mitochondria was slightly enhanced following hormone treatment, but not the basal or the total ATPase activity measured in the presence of Triton X-100 plus Mg2+. The mitochondria prepared from dexamethasone-treated rats also displayed increased intramitochondrial concentrations of Mg2+, K+ and exchangeable adenine nucleotides but not Ca2+. It is suggested that the effect of glucocorticoids on mitochondrial respiration may be both the result of a direct activation of the respiratory chain within Complex III and an elevated intramitochondrial adenine nucleotide concentration. The evidence for the de novo synthesis of mitochondrial proteins which mediate the response remains inconclusive.     

The possible role of palmitoyl-CoA in the regulation of the adenine nucleotides transport in mitochondria under different metabolic states. I. Comparison of liver mitochondria from starved and fed rats.

It has been shown that KM values for ADP when rat liver mitochondria oxidized succinate were strictly dependent on the values of the respiratory control ratios. The Ki values for palmitoyl-CoA inhibition of the ADP-stimulated succinate oxidation and the inhibition of the uncoupler-stimulated ATPase activity were equal to 0.5 muM. Mitochondria from livers of starved rats showed 30% inhibition of the state 3 respiratory rate (compared to the uncoupled respiratory rate) which was abolished by addition of carnitine. It was supposed that this inhibition was due to the influence of acyl-CoAs bound to the inner mitochondrial membrane on the adeninenucleotide translocase. Mitochondria from livers of fed rats showed a strong inhibition of succinate oxidation both in state 4 and state 3, although the rate of uncoupled respiration was normal. It was assumed that in this case the changes in mitochondrial behaviour was caused by the decrease in the concentration of ADP and ATP in the matrix space of mitochondria.     

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 (≥0.4 μM). 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.     

Differential alterations in mitochondrial function induced by a choline-deficient diet: understanding fatty liver disease progression

Non-alcoholic fatty liver disease (NAFLD) is an increasingly reported pathology, characterized by fat accumulation within the hepatocyte. Growing evidences suggest specific effects on mitochondrial metabolism, but it is still unclear the relationship between fatty liver progression and mitochondrial function. In the present work we have investigated the impact of fatty liver on mitochondrial bioenergetic functions and susceptibility to mitochondrial permeability transition (MPT) induction in animals fed a choline-deficient diet (CDD) for 4, 8, 12 or 16 weeks. Mitochondria isolated from CDD animals always exhibited higher state 4 respiration. Mitochondrial membrane potential was decreased in CDD animals at 4 and 16 weeks. At 12 weeks, oxidative phosphorylation was more efficient in CDD animals, suggesting a possible early response trying to revert the deleterious effect of increased triglyceride storage in the liver. However, mitochondrial dysfunction was evident in CDD animals at 16 weeks as indicated by decreased RCR and ADP/O, with a corresponding decrease in respiratory chain enzymes activities. Such loss of respiratory efficiency was associated with accumulation of protein oxidation products, in tissue and mitochondrial fraction. Additionally, although no differences in ATPase activity, the lag phase was increased in mitochondria from CDD animals at 16 weeks, associated with decreased content of the adenine nucleotide translocator. Increased susceptibility to calcium-induced MPT was evident in CDD animals at all time points. These results suggest a dynamic mechanism for the development of NALFD associated with altered mitochondrial function.     

Selective Impairment of Respiration in Mitochondria Isolated from Brain Subregions Following Transient Forebrain Ischemia in the Rat

Using Percoll density gradient centrifugation, free (nonsynaptosomal) mitochondria were isolated from the dorsal-lateral striatum and paramedian neocortex of rats during complete forebrain ischemia and reperfusion. Mitochondria prepared from either region after 30 min of ischemia showed decreased state 3 (ADP and substrate present) and uncoupled respiration rates (19-45% reductions) with pyruvate plus malate as substrates, whereas state 4 respiration (no ADP present) was preserved. At 6 h of recirculation, state 3 and uncoupled respiration rates for mitochondria from the paramedian neocortex (a region resistant to ischemic damage) were similar to or even increased compared with control values. By contrast, in mitochondria from the dorsal-lateral striatum (a region containing neurons susceptible to global ischemia), decreases in state 3 and uncoupled respiration rates (25 and 30% less than control values) were again observed after 6 h of recirculation. With succinate as respiratory substrate, however, no significant differences from control values were found in either region at this time point. By 24 h of recirculation, respiratory activity with either pyruvate plus malate or succinate was greatly reduced in samples from the dorsal-lateral striatum, probably reflecting complete loss of function in some organelles. In contrast with these marked changes in free mitochondria, the respiratory properties of synaptosomal mitochondria, assessed from measurements in unfractionated homogenates, were unchanged from controls in the dorsal-lateral striatum at each of the time points studied, but showed reductions (19-22%) during ischemia and after 24 h of recirculation in the paramedian neocortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

A possible mechanism of mitochondrial dysfunction during cerebral ischemia: inhibition of mitochondrial respiration activity by arachidonic acid.

The dramatic increase in the arachidonic acid (AA) level in the brain is a well-known molecular event during cerebral ischemia. As mitochondria are known to be one possible site of the cell damage, the effects of AA on the respiratory activity of rat brain mitochondria were investigated in vitro using an oxygen electrode. In NAD-linked respiration, respiratory control ratio was decreased significantly by AA, with an IC50 of 6.0 microM. AA had the dual effect on mitochondrial respiration, a decrease in state 3 and uncoupled state and an increase in state 4 (i.e., uncoupling) as reported by Hillered and Chan (J. Neurosci. Res. 19, 94-100, 1988). Furthermore, we found that other unsaturated long-chain free fatty acids (C18:1-C18:3, C20:1-C20:5) also showed such a dual effect. Cyclooxygenase metabolites of AA such as prostaglandins (D2, E2, F2 alpha, E1) and thromboxane B2, and lipoxygenase metabolites such as leukotrienes (D4, B4) and 5- or 12-hydroperoxyeicosatetraenoic acid had no significant effect. The inhibition of the uncoupled state by AA was more marked in NAD-linked than that in FAD-linked respiration, while the degree of uncoupling by AA were the same in both respirations. In spectrophotometrical measurement, the reduction of cytochromes and flavo-protein was markedly inhibited by AA in NAD-linked respiration, but not in the FAD-linked one. In addition, the activity of cytochrome c oxidase was scarcely inhibited by AA. These data suggest that AA itself, not its metabolites, may inhibit mitochondrial ATP production during brain ischemia and that AA may act on the site(s) closely related to NAD-linked respiration, but not the FAD-linked one, in addition to its uncoupling effect.     

Arachidonic acid interaction with the mitochondrial electron transport chain promotes reactive oxygen species generation.

A study has been carried out on the interaction of arachidonic acid and other long chain free fatty acids with bovine heart mitochondria. It is shown that arachidonic acid causes an uncoupling effect under state 4 respiration of intact mitochondria as well as a marked inhibition of uncoupled respiration. While, under our conditions, the uncoupling effect is independent of the fatty acid species considered, the inhibition is stronger for unsaturated acids. Experiments carried out with mitochondrial particles indicated that the arachidonic acid dependent decrease of the respiratory activity is caused by a selective inhibition of Complex I and III. It is also shown that arachidonic acid causes a remarkable increase of hydrogen peroxide production when added to mitochondria respiring with either pyruvate+malate or succinate as substrate. The production of reactive oxygen species (ROS) at the coupling site II was almost double than that at site I. The results obtained are discussed with regard to the impairment of the mitochondrial respiratory activity as occurring during the heart ischemia/reperfusion process.     

Effect of Peroxide, Sodium, and Calcium on Brain Mitochondrial Respiration In Vitro: Potential Role in Cerebral Ischemia and Reperfusion

Mitochondrial pyruvate-supported respiration was studied in vitro under conditions known to exist following ischemia, i.e., elevated extramitochondrial Ca2+, Na+, and peroxide. Ca2+ alone (7-10 nmol/mg) decreased state 3 and increased state 4 respiration to 81 and 141% of control values, respectively. Sodium (15 mM) and/or tert-butyl hydroperoxide (tBOOH; up to 2,000 nmol/mg protein) alone had no effect on respiration; however, Na+ or tBOOH in combination with Ca2+ dramatically altered respiration. Respiratory inhibition induced by Ca2+ and tBOOH does not involve pyruvate dehydrogenase (PDH) inhibition since PDH flux increased linearly with tBOOH concentration (R = 0.96). Calcium potentiated tBOOH-induced mitochondrial NAD(P)H oxidation and shifted the redox state of cytochrome b from 67 to 47% reduced. Calcium (5.5 nmol/mg) plus Na+ (15 mM) decreased state 3 and increased state 4 respiratory rates to 55 and 202% of control values, respectively. Sodium- as well as tBOOH-induced state 3 inhibition required mitochondrial Ca2+ uptake because ruthenium red addition before Ca2+ addition negated the effect. The increase in state 4 respiration involved Ca2+ cycling since ruthenium red immediately returned state 4 rates back to control values. The mechanisms for the observed Ca2(+)-, Na(+)-, and tBOOH-induced alterations in pyruvate-supported respiration in vitro are discussed and a multifactorial etiology for mitochondrial respiratory dysfunction following cerebral ischemia in vivo is proposed.     

ORGANIC MERCURIAL ENCEPHALOPATHY: IN VIVO AND IN VITRO EFFECTS OF METHYL MERCURY ON SYNAPTOSOMAL RESPIRATION

—A reproducible model of subacute methyl mercury (MeHg) intoxication was developed in the adult rat following the daily intragastric administration of 10 mg methyl mercury/kg body wt. Synaptosomes isolated from animals during the latent phase of mercury neurotoxicity (6-10 days) demonstrated no significant change in respiratory control, State 3, State 4, or 2,4-dinitrophenol stimulated respiration with succinate, glutamate or pyruvate plus malate. During the neurotoxic phase, a significant decline in respiratory control was evident with all substrates. Cerebellar synaptosomes revealed qualitatively similar but quantitatively greater inhibition of 2,4-dinitrophenol stimulated respiration during the latent and neurotoxic phases with glutamate. In vitro studies of synaptosome respiration, oxidative phosphorylation and respiratory control with 5-15 μm -methyl mercury revealed a stimulation of initial State 4 respiration, loss of RCI, inhibition of State 3 but no change in the gramicidin or 2,4-dinitrophenol uncoupled rate supported by pyruvate-malate. Phosphate did not relieve the State 3 inhibition. At 25 μm -methyl mercury and above, considerable inhibition of electron transfer occurred. At this concentration, cytochrome c oxidase was inhibited 50%. Isosmotic replacement of medium KC1 by mannitol reduced the MeHg stimulation of State 4 respiration but had no effect on MeHg inhibition of ADP stimulated respiration. Half-maximal stimulation of State 4 respiration by MeHg occurred at [K]⁺⋍ 6 mm . These findings are compatible with an energy-linked methyl mercury induced cation translocation across the synaptosome (mitochondrial) membrane.     

Chromium(VI) interaction with plant and animal mitochondrial bioenergetics: a comparative study

The mechanism of Cr(VI)-induced toxicity in plants and animals has been assessed for mitochondrial bioenergetics and membrane damage in turnip root and rat liver mitochondria. By using succinate as the respiratory substrate, ADP/O and respiratory control ratio (RCR) were depressed as a function of Cr(VI) concentration. State 3 and uncoupled respiration were also depressed by Cr(VI). Rat mitochondria revealed a higher sensitivity to Cr(VI), as compared to turnip mitochondria. Rat mitochondrial state 4 respiration rate triplicated in contrast to negligible stimulation of turnip state 4 respiration. Chromium(VI) inhibited the activity of the NADH-ubiquinone oxidoreductase (complex I) from rat liver mitochondria and succinate-dehydrogenases (complex II) from plant and animal mitochondria. In rat liver mitochondria, complex I was more sensitive to Cr(VI) than complex II. The activity of cytochrome c oxidase (complex IV) was not sensitive to Cr(VI). Unique for plant mitochondria, exogenous NADH uncoupled respiration was unaffected by Cr(VI), indicating that the NADH dehydrogenase of the outer leaflet of the plant inner membrane, in addition to complexes III and IV, were insensitive to Cr(VI). The ATPase activity (complex V) was stimulated in rat liver mitochondria, but inhibited in turnip root mitochondria. In both, turnip and rat mitochondria, Cr(VI) depressed mitochondrial succinate-dependent transmembrane potential (Deltapsi) and phosphorylation efficiency, but it neither affected mitochondrial membrane permeabilization to protons (H+) nor induced membrane lipid peroxidation. However, Cr(VI) induced mitochondrial membrane permeabilization to K+, an effect that was more pronounced in turnip root than in rat liver mitochondria. In conclusion, Cr(VI)-induced perturbations of mitochondrial bioenergetics compromises energy-dependent biochemical processes and, therefore, may contribute to the basal mechanism underlying its toxic effects in plant and animal cells.     

Delta sleep inducing peptide (DSIP): effect on respiration activity in rat brain mitochondria and stress protective potency under experimental hypoxia

Neuromodulatory delta sleep inducing peptide (DSIP) seems to be implicated in the attenuation of stress-induced pathological metabolic disturbances in various animal species and human beings. Mitochondria, as cell organelles, are considered especially sensitive to stress conditions. In this work, the influence of DSIP and Deltaran((R))-a recently developed product based upon DSIP-on processes of oxidative phosphorylation and ATP production in rat brain mitochondria and rat brain homogenates was studied. A polarographic measurement of oxygen consumption was applied to evaluate the impact of DSIP on maximal rates of mitochondrial respiration and coupling of respiration to ATP production. We provide evidence that DSIP affected the efficiency of oxidative phosphorylation on isolated rat brain mitochondria. This peptide significantly increased the rate of phosphorylated respiration V3, while the rate of uncoupled respiration V(DNP) remaining unchanged. It enhanced the respiratory control ratio RCR and the rate of ADP phosphorylation. DSIP and Deltaran exhibited the same action in rat brain homogenates. We also examined the influence of DSIP under hypoxia when mitochondrial respiratory activity is altered. In rats subjected to hypoxia, we detected a significant stress-mediated reduction of V3 and ADP/t values. Pretreatment of rats with DSIP at the dose of 120 microgram/kg (i.p.) prior to their subjection to hypoxia completely inhibited hypoxia-induced reduction of mitochondrial respiratory activity. The revealed capacity of DSIP to enhance the efficiency of oxidative phosphorylation found in vitro experiments could contribute to understanding pronounced stress protective and antioxidant action of this peptide in vivo.     

Effect of Iron Deficiency on the Respiration of Sycamore (Acer pseudoplatanus L.) Cells

The effects of iron deficiency on cell culture growth, cell respiration, mitochondrial oxidative properties, and the electron transport chain were studied with suspension-cultured sycamore (Acer pseudoplatanus L.) cells. Iron deprivation considerably decreased the initial growth rates and limited the maximum density of the cells. Under these conditions, the cells remained swollen throughout their growth. The absence of iron led to a steady decline in the uncoupled rate of O2 consumption. When the uncoupled rate of O2 uptake closely approximated the respiratory rate, the cells began to collapse. At this stage, the level of all the cytochromes and electron paramagnetic resonance-detectable Fe-S clusters of the mitochondrial inner membrane were dramatically decreased. Nevertheless, it appeared from substrate oxidation measurements that this overall depletion in iron-containing components solely disturbed the functioning of complex II, whereas neither complexes I, III, or IV, nor the machinery involved in ATP synthesis, was apparently impaired in iron-deficient mitochondria. However, our results suggest that the impairment of complex II resulted in a strong reduction of the overall capacity of the mitochondrial electron transport chain, which was responsible for determining the rate of endogenous respiration in sycamore cells. Finally, this situation led to a depletion of various energy metabolites that could contribute to the premature cell death.     

Oxidative phosphorylation in mitochondria isolated from small intestinal epithelium of thiamphenicol-treated rats and control rats

Treatment of rats with thiamphenicol in a dose of 125 mg/kg per day for 60–64 h causes specific inhibition of mitochondrial protein synthesis, leading to a drastic decrease of the cytochrome c oxidase activity in intestinal epithelium. At the same time the mitochondrial ATPase activity becomes resistant to inhibition by oligomycin. Experiments with isolated intestinal mitochondria revealed that respiration in state 3 is diminished by 55% with succinate (5 mM) and by 40% with pyruvate (10 mM) plus L-malate (2 mM) as the substrates, both as compared to intestinal mitochondria isolated from control rats. P : O ratios as well as respiratory control indices are comparable in the two groups of animals. Uncoupled respiration is inhibited by 35% with succinate as the substrate, while the succinate cytochrome c reductase activity remains unaltered. No inhibition of uncoupled respiration with pyruvate plus L-malate as the substrates was observed. The ATP-P_i exchange activity in the mitochondria from the treated animals is diminished by about 75%. It is concluded that in the mitochondria of the treated animals the inhibition of the coupled respiration (state 3) is caused by the limitation of the ATP-generating capacity and that electron transport is rate limiting only with the rapidly oxidized substrates such as succinate, if respiration is uncoupled.     

Effect of heliomycin on the respiration and oxidative phosphorylation of the liver mitochondria of the rat

The effect of heliomycin and known uncouplers of oxidative phosphorylation on respiration and oxidative phosphorylation was studied comparatively. Heliomycin, as well as 2,4-dinitrophenol, valinomycin and gramicidin S inhibited the mitochondrial synthesis of ATP. This process was inhibited completely by heliomycin at a concentration of 1.5 x 10(-5) M. The synthesis of inorganic pyrophosphate, the other macroergic compound, was also inhibited by heliomycin, ATPase and pyrophosphatase of uncoupled mitochondria being not inhibited by the antibiotic. Like 2,4-dinitrophenol, heliomycin stimulated the synthesis of ATPase and respiration in intact mitochondria. Probably, heliomycin inhibited the synthesis of ATP and pyrophosphate by uncoupling the processes of respiration and oxidative phosphorylation. It was shown earlier that heliomycin, a specific inhibitor of bacterial RNA synthesis, also affected energy metabolism of bacterial cells by inhibiting the synthesis of ATP and active transport.     

Site of Action of the Cord Factor of Corynebacterium diphtheriae in Mitochondria

Trehalose-6,6'-dicorynomycolate (T66DCM), the cord factor of Corynebacterium diphtheriae, induced in vitro a swelling accompanied with a partially irreversible change of the mitochondrial membrane system in mouse liver. Preincubation of the mitochondrial suspension with T66DCM resulted in an inhibition of phosphorylation coupled to the oxidation of either succinate or a number of reduced nicotinamide adenine dinucleotide-linked substrates and a loss of respiratory control. T66DCM affected both electron transport and phosphorylation at coupling site II and uncoupled respiration and phosphorylation at coupling site III. T66DCM stimulated mitochondrial adenosine triphosphatase. The induction of adenosine triphosphatase by T66DCM and by 2,4-dinitrophenol was additive.     

Effects of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) on mitochondrial bioenergetics and oxidative stress: a comparative study

The potential protective action of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) against oxidative stress was assessed on mitochondrial bioenergetics, inner membrane anion channel (IMAC), Ca2+-induced opening of the permeability transition pore (PTP), and oxidative damage induced by the oxidant pair adenosine diphosphate (ADP)/Fe2+ (lipid peroxidation) of mitochondria isolated from rat liver. By using succinate as the respiratory substrate, respiratory control ratio (RCR), ADP to oxygen ratio (ADP/O), state 3, state 4, and uncoupled respiration rates were not significantly affected by gammapyrone, glutapyrone, and diethone concentrations up to 100 microM. Cerebrocrast at concentrations higher than 25 microM depressed RCR, ADP/O, state 3, and uncoupled respiration rates, but increased three times state 4 respiration rate. The transmembrane potential (deltapsi) and the phosphate carrier rate were also decreased. At concentrations lower than 25 microM, cerebrocrast inhibited the mitochondrial IMAC and partially prevented Ca2+-induced opening of the mitochondrial PTP, whereas gammapyrone, glutapyrone, and diethone were without effect. Cerebrocrast, gammapyrone, and glutapyrone concentrations up to 100 microM did not affect ADP/Fe2+-induced lipid peroxidation of rat liver mitochondria, while very low diethone concentrations (up to 5 microM) inhibited it in a dose-dependent manner, as measured by oxygen consumption and thiobarbituric acid reactive substances formation. Diethone also prevented deltapsi dissipation due to lipid peroxidation initiated by ADP/Fe2+. It can be concluded that: none of the compounds interfere with mitochondrial bioenergetics at concentrations lower than 25 microM; cerebrocrast was the only compound that affected mitochondrial bioenergetics, but only for concentrations higher than 25 microM; at concentrations that did not affect mitochondrial bioenergetics (< or = 25 microM), only cerebrocrast inhibited the IMAC and partially prevented Ca2+-induced opening of the PTP; diethone was the only compound that expressed antioxidant activity at very low concentrations (< or = 5 microM). Cerebrocrast acting as an inhibitor of the IMAC and diethone acting as an antioxidant could provide effective protective roles in preventing mitochondria from oxidative damage, favoring their therapeutic interest in the treatment of several pathological situations known to be associated with cellular oxidative stress.     

Acutely administered melatonin restores hepatic mitochondrial physiology in old mice

Damage to mitochondria as a result of the intrinsic generation of free radicals is theoretically involved in the processes of cellular aging. Herein, we investigated whether acutely administered melatonin, due to its free radical scavenging activity, would influence mitochondrial metabolism. Mitochondrial respiratory activity and respiratory chain complex I and IV activities in liver mitochondria from a strain of senescence-accelerated-prone mice (SAMP8) and a strain of senescence-accelerated-resistant mice (SAMR1) were measured when the animals were 12 months of age. Respiratory control index (RCI), ADP/O ratio, State 3 respiration and dinitrophenol (DNP)-dependent uncoupled respiration were significantly lower in SAMP8 than in SAMR1. In contrast, State 4 respiration was significantly higher in SAMP8 than in SAMR1. Activities of complexes I and IV in SAMP8 were significantly lower than in SAMR1. Melatonin administration (10mg/kg body weight, intraperitoneally) 1h prior to sacrifice significantly increased RCI, ADP/O ratio, State 3 respiration and DNP-induced uncoupled respiration in SAMP8 while also significantly reducing State 4 respiration in SAMP8. The injection of melatonin also significantly increased complex I activity in both mouse strains and complex IV activity in the liver of SAMP8 mice. These results document an age-related decrease in hepatic mitochondrial function in SAM which can be modified by an acute pharmacological injection of melatonin; the indole stimulated mitochondrial respiratory chain activity which would likely reduce deteriorative oxidative changes in mitochondria that normally occur in advanced age.