Protective effects of resveratrol on calcium-induced oxidative stress in rat heart mitochondria

Trans-resveratrol is a nutraceutical with known antioxidant, anti-inflammatory, cardioprotective, and anti-apoptotic properties. The aim of this study was to evaluate the effects of resveratrol on heart mitochondria. Resveratrol significantly decreased Fe²⁺ + ascorbate oxidant system-induced lipid peroxide levels, preserved physiological levels of glutathione, and increased nitric oxide (NO) levels in mitochondria. Under calcium-mediated stress, there was a 2.7-fold increase in the NO levels, and a mild decoupling in the mitochondrial respiratory chain. These results provide a mechanism for and support the beneficial effects of resveratrol under pathological conditions induced by oxidative stress and calcium overload. In addition, these findings underscore the usefulness of resveratrol in the prevention of cardiovascular diseases.     

Fumarate permeation in rat heart mitochondria

The possible fumarate translocation in rat heart mitochondria is examined. This substrate, which is claimed to be a non permeant ion in rat liver mitochondria appears to cross the mitochondrial membrane in cardiac mitochondria. This conclusion was proposed on the basis of experimental results which show swelling by rat heart mitochondria in ammonium fumarate, uptake by mitochondria of fumarate, Pi efflux from the matrix induced by fumarate and appearance of malate in the reaction mixture which follows the addition of fumarate to the mitochondria and depends on the fumarase activity. The existence of a carrier unknown so far as well as a possible physiological role of this transport is proposed.     

Studies on the mechanism of the inhibitory effects of erucylcarnitine in rat heart mitochondria.

1. The mechanism of the inhibitory effect of erucylcarnitine on palmityl-carnitine oxidation in rat heart mitochondria was studied. 2. Erucylcarnitine inhibited in the same time the oxidation of [U-14-C]-palmitylcarnitine and the total rate of oxygen uptake. Other acylcarnitines competed as well for the oxidation with radioactive palmitylcarnitine, but they were well oxidized themselves, so that the total oxygen uptake did not decrease. 3. The presence of erucylcarnitine did not change the distribution pattern of Krebs cycle intermediates derived from [U-minus 14 C] palmitylcarnitine except that succinate/malate ratio increased. 4. The presence of erucylcarnitine did not lead to the formation of any beta-oxidation cycle intermediates from [U-minus 14 C] palymitylcarnitine. The formation of beta-hydroxy-palmityl derivative when rotenon was included into the incubation medium, decreased in the presence of erucylcarnitine. 5. It is postulated, that the inhibited entrance of palmityl groups into the beta-oxidation cycle is due to the fact that erucylcarnitine and palmitylcarnitine behave as substrate-competitive inhibitors for long chain acyl-CoA dehydrogenase. 6. There was observed a latency of 1-2 min in the effect of erucylcarnitine on the palmitylcarnitine oxidation, which seems to correspond to the time required for the formation of high amounts of intramitochondrial erucyl-CoA. 7. Erucylcarnitine inhibited the total oxygen uptake with long, medium and short chain acylcarnitines, pyruvate and alpha-ketoglutarate as substrates, while the oxidation of succinate was not affected. 8. Sequestration of free CoA in the form of very slowly metabolized erucyl-CoA is proposed as the partial explanation of the observed inhibitory effects of erucylcarnitine on the oxidation of CoA-dependent substrates (alternatively to the inhibition at the level of acyl-CoA dehydrogenases in case of acylcarnitines).     

Uncoupled oxidation in rat heart mitochondria

Isolated heart mitochondria possessing a high phosphorylation efficiency with pyruvate and malate as substrates oxidize NADH and ascorbate unassociated with ADP phosphorylation. This uncoupled pathway is expressed partially when succinate or NAD-linked substrates are oxidized. The uncoupled oxidation is likely to be the result of the presence of a mitochondrial population with the high-permeable inner membrane in intact tissues. The nature and origin of a uncoupled respiratory system and its role in the thermoproduction of endotherms are discussed.     

Studies of adenosine incorporation in Langendorff rat heart and rat heart mitochondria

The acid-insoluble product isolated from well-oxygenated Langendorff rat heart after perfusion with [¹⁴C]adenosine was purified by phenol extraction and subjected to specific phosphorolysis by pure polynucleotide phosphorylase. TLC analysis of the reaction mixture showed that ADP was the only radioactive product, proving that the original substance was a polyribonucleotide. Studies of the time course of labelling and of the distribution of the acid-insoluble product between the mitochondrial and nuclear fractions showed that both are labelled even after 1 min at 25 °C, but at short times and low temperature more radioactivity is found in the mitochondria. The kinetics of adenosine incorporation resemble those expected for the labelling of hnRNA and mRNA. Isolated, respiring mitochondria incorporate adenosine and adenine nucleotides into acid insoluble form by a process dependent on oxidative phosphorylation and the adenine nucleotide translocase that is specific for adenine derivatives. The results are discussed in terms of the hypothesis that the polyribonucleotide might be a storage form of adenine nucleotides: it is concluded that the bulk of the labelled product is unlikely to play a major role in energy metabolism.     

Some effects of decenylsuccinic Acid on isolated corn mitochondria

The effects of decenylsuccinic acid on the swelling and respiratory capacities of mitochondria isolated from etiolated corn (Zea mays L., Wf9 × M14) shoots were studied. Decenylsuccinic acid (0.1 mM to 1.0 mM) inhibited the oxidation of succinate and malate-pyruvate, stimulated the oxidation of reduced nicotinamide adenine dinucleotide, and uncoupled phosphorylation. The swelling of isolated corn mitochondria, as determined by percentage of transmittance changes, was stimulated by decenylsuccinic acid in potassium chloride reaction media and in sucrose reaction media without bovine serum albumin. In a diaphorase (2, 6-dichlorophenolindophenol as acceptor) reaction with intact mitochondria, only the dehydrogenation rate of malate was reduced by the addition of decenylsuccinic acid. The dehydrogenation of reduced nicotinamide adenine dinucleotide or of succinate was either not affected or was stimulated depending on the diaphorase reaction medium. The oxygen uptake of mitochondria oxidizing N, N, N′, N′-tetramethyl-p-phenylenediamine diHCl and ascorbate was inhibited at decenylsuccinic acid concentrations greater than 0.5 mM.     

A comparative study of the effects of procaine, lidocaine, tetracaine and dibucaine on the functions and ultrastructure of isolated rat liver mitochondria

The effects of procaine, lidocaine, tetracaine and dibucaine (10(-5) - 10(-2) M) were tested on isolated rat liver mitochondria by measurements of the respiratory rates and of the membrane potential and by electron microscopy. A general concentration-dependent stimulation of the basal state (respiration before ADP addition) was observed for all local anesthetics studied. Up to the concentration of 10(-3) M, the order of stimulation was: procaine less than lidocaine less than dibucaine less than tetracaine. However, with the exception of dibucaine, which inhibited state-3 respiration (ADP present) in a strictly concentration-dependent manner, the other drugs had a biphasic effect: slight stimulation of state 3 at low and moderate concentrations (less than or equal to 10(-3) M) and inhibition at higher concentrations. Nevertheless, due to a stronger stimulation of the basal state, the acceptor control ratio decreases progressively (uncoupling effect) as the concentration of the drugs increases. The only exception to this observation is procaine in the range of 10(-5) - 10(-4) M, where the stimulation of the two respiration states (although small) is approximately equal and thus the uncoupling effect is absent or negligible. Membrane potential recordings suggested that membrane integrity and phosphorylation capacity were negatively affected at high drug concentrations (greater than 10(-3) M), especially in the case of tetracaine and dibucaine, when 5 x 10(-3) M even produced the collapse of the membrane potential and complete loss of the phosphorylation ability. Electron microscopy confirmed these effects, showing an abundance of either swollen or supercondensed mitochondria, with many membrane ruptures. The action mechanisms of the tertiary amines studied are discussed in terms of interaction of drug with the lipid bilayer and with the membrane proteins. It is concluded that both the inhibitory and the uncoupling effects are dependent, in the first place, on the degree of hydrophobicity of each local anesthetic.     

Kynurenines and the respiratory parameters on rat heart mitochondria

It has been shown recently that the L-kynurenine metabolite kynurenic acid lowers the efficacy of mitochondria ATP synthesis by significantly increasing state IV, and reducing respiratory control index and ADP/oxygen ratio of glutamate/malate-consuming heart mitochondria. In the present study we investigated the effect of L-tryptophan (1.25 microM to 5 mM) and other metabolites of L-kynurenine as 3-hydroxykynurenine (1.25 microM to 2.5 mM), anthranilic acid (1.25 microM to 5 mM) and 3-hydroxyanthranilic acid (1.25 microM to 5 mM) on the heart mitochondria function. Mitochondria were incubated with saturating concentrations of respiratory substrates glutamate/malate (5 mM), succinate (10 mM) or NADH (1 mM) in the presence or absence of L-tryptophan metabolites. Among tested substances, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and anthranilic acid but not tryptophan affected the respiratory parameters dose-dependently, however at a high concentration, of a micro molar range. 3-Hydroxykynurenine and 3-hydroxyanthranilic acid lowered respiratory control index and ADP/oxygen ratio in the presence of glutamate/malate and succinate but not with NADH. While, anthranilic acid reduced state III oxygen consumption rate and lowered the respiratory control index only of glutamate/malate-consuming heart mitochondria. Co-application of anthranilic acid and kynurenic acid (125 or 625 microM each) to glutamate/malate-consuming heart mitochondria caused a non-additive deterioration of the respiratory parameters determined predominantly by kynurenic acid. Accumulated data indicate that within L-tryptophan metabolites kynurenic acid is the most effective, followed by anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid to influence the respiratory parameters of heart mitochondria. Present data allow to speculate that changes of kynurenic acid and/or anthranilic acid formation in heart tissue mitochondria due to fluctuation of L-kynurenine metabolism may be of functional importance for cardiovascular processes. On the other hand, beside the effect of 3-hydroxyanthranilic acid and 3-hydroxykynurenine on respiratory parameters, their oxidative reactivity may contribute to impairment of mitochondria function, too.     

Independent and additive effects of atenolol and methionine restriction on lowering rat heart mitochondria oxidative stress

A low rate of mitochondrial ROS production (mitROSp) and a low degree of fatty acid unsaturation are characteristic traits of long-lived animals and can be obtained in a single species by methionine restriction (MetR) or atenolol (AT) treatments. However, simultaneous application of both treatments has never been performed. In the present investigation it is shown that MetR lowers mitROSp and complex I content. Both the MetR and the AT treatments lower protein oxidative modification and oxidative damage to mtDNA and the fatty acid unsaturation degree in rat heart mitochondria. The decrease in fatty acid unsaturation seems to be due, at least in part, to decreases in desaturase and elongase activities or peroxisomal β-oxidation. Furthermore, the phosphorylation of extracellular signal-regulated kinase (ERK) was stimulated by MetR and AT. The decrease in membrane fatty acid unsaturation and protein oxidation, and the changes in fatty acids and p-ERK showed additive effects of both treatments. In addition, the increase in mitROSp induced by AT observed in the present investigation was totally avoided with the combined MetR + AT treatment. It is concluded that the simultaneous treatment with MetR plus atenolol is more beneficial than either single treatment alone to lower oxidative stress in rat heart mitochondria, analogously to what has been reported in long-lived animal species.     

Anisotropic action of cetyl pyridinium chloride on rat heart mitochondria

This work reports experiments that show that in rat heart mitochondria, the alkyl cation cetyl pyridinium chloride induces inhibition of the electron transport with NAD-dependent substrates. It also induces an enhancement of oxygen uptake with succinate as substrate, stimulation of adenosine triphosphatase activity, release of Ca²⁺ that have been accumulated, and inhibition of the energy-dependent uptake of ethidium bromide; these findings suggest that cetyl pyridinium chloride induces a collapse of membrane potential. The experiments carried out with submitochondrial particles showed that this reagent inhibits the oxidation of NADH, provided an uncoupler is added to the system. According to these data it is proposed that the latter effect is due to the binding of cetyl pyridinium chloride to the inner mitochondrial membrane in a site that faces the cytosol.     

Anion-sensitive ATPase of the rat heart mitochondria]

The properties of anion-sensitive ATPase of rat heart mitochondria were studied. Na2CO3, NaHCO3 and Na2SO3 stimualted ATPase activity by 69, 41 and 110%, respectively. Azide, tiocinate and perchlorate inhibited bicarbonate-stimulated ATPase. Bivalent cations increased ATPase activity in such a sequence: Zn2+ greater than or equal to Cd2+ greater than or equal to Co2+ greater than or equal to Mg2+ greater than or equal to Mn2+ greater than Ni2+. In the presence of bicarbonate and sulfite. ATPase activity was maximally stimulated with magnesium. Ni2+ and Ca2+-ions inhibited Mg2+-dependent activity of bicarbonate-stimulated ATPase. AMP uninhibited ATPase activity. The 4 mM concentration of ADP inhibited activity of HCO-3-ATPase. Activity of ATPases decreased at lower temperatures. The properties of anion-sensitive ATPase of rat heart mitochondria and that of HCO-2-ATPase of other cells are discussed.