Stobadine inhibits lysosomal enzyme release in vivo and in vitro

This study investigated the ability of stobadine, an effective cardioprotective drug with antiarrhythmic, antihypoxic and oxygen free radical scavenging properties, to protect cells against cyclophosphamide-induced toxic and cytotoxic damage in vivo and in vitro. Cyclophosphamide-induced toxic damage in female ICR mice was accompanied by marked increase in the activity of lysosomal enzymes in the spleen and kidney. Administration of stobadine prior to cyclophosphamide inhibited these biochemical changes. The in vivo protective effect of stobadine was comparable with its in vitro effect established in HeLa cells.     

Ion transport by heart mitochondria. Retention and loss of energy coupling in aged heart mitochondria

The retention and loss of energy-coupling reactions in isolated beef heart mitochondria have been examined under anaerobic conditions using suspending media chosen to mimic the intracellular milieu. In long-term incubations at 37 °C, a loose coupling develops which can be controlled by adding serum albumin. This lesion closely resembles that produced by addition of free fatty acids which has been described in previous studies. Shorter incubation times produce an increased susceptibility to hydrogen peroxide which is characterized by elevated ATPase activity, increased permeability to monovalent cations, and increased proton ejection on transition from the anaerobic to the aerobic state. This peroxide sensitivity is prevented by chelators such as EGTA and appears to involve a time-dependent release of metal ions. Of the metabolites which are known to increase in concentration in the ischemic heart cell, Na⁺, P₁, lactate, and H⁺ all promote swelling of isolated heart mitochondria and contribute to a decline in energy coupling. The relationship of these results to the pathological deterioration of mitochondria in ischemic heart tissue is discussed.     

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.     

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.     

Release of AMP and adenosine from rat heart mitochondria

Release of AMP and adenosine from rat heart mitochondria was studied. The rate of appearance of extramitochondrial adenosine was independent of the extramitochondrial phosphate concentration between 5 and 20 mM. In the absence of exogenous, respiratory substrates or in the presence of glutamate/malate plus rotenone, the rate of appearance of adenosine was relatively low when phosphate was not added. The appearance of extramitochondrial AMP + adenosine was found to be directly proportional to the extra-mitochondrial phosphate concentration. Zn2+ (10 mM) decreased the rate of adenosine appearance by 90% and increased the rate of AMP appearance 6-fold. The mitochondrial preparations dephosphorylated exogenous AMP; this activity was inhibited by 10 mM Zn2+. We conclude that the adenosine appearing in the extramitochondrial space was not due to a direct release from the matrix, but instead was due to adenine nucleotide release with subsequent conversion to adenosine in the extramitochondrial space.     

Lipids and lipolytic enzyme activities of rat heart mitochondria

The lipid composition and lipolytic enzyme activities in rat cardiac mitochondria were examined. Subsarcolemmal mitochondria were prepared by treatment of heart muscle with a Polytron tissue processor, while interfibrillar mitochondria were released by exposure of the remaining low-speed pellet to the protease, nagarse. These procedures are known to yield two functionally different populations of mitochondria. However, their phospholipid contents and compositions were identical, as were the positional distributions of the constituent fatty acids. Of the ethanolamine phospholipids, 20% were plasmalogens, and about 2% of the choline phospholipids consisted of this alkenylacyl species. Both subsarcolemmal and interfibrillar mitochondria contained a Ca²⁺-activated phospholipase A₂, as evidenced by the Ca²⁺-dependent release of unsaturated fatty acids and lysophosphatidylethanolamine from endogenous lipids. Ruthenium red prevented the activation of this enzyme by Ca²⁺, indicating that the activity is located in the matrix space or associated with the inner surface of the inner membrane. Both mitochondrial fractions produced free fatty acids and lysophosphatidylethanolamine in the absence of free Ca²⁺ apparently due to an outer membrane phospholipase A₁. The activity of this enzyme decreased with time, particularly in interfibrillar mitochondria, providing that Ca²⁺ was absent. Nagarse treatment of subsarcolemmal mitochondria resulted in a preparation with the same phospholipase A₁ properties as interfibrillar mitochondria. The possibility that differences in phospholipase A₁ properties account for some of the functional variations between the two mitochondrial types is discussed.     

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.     

Oscillatory states of mitochondria. Studies on the oscillatory mechanism of liver and heart mitochondria

Oscillatory movements of ions across the inner membrane of liver and heart mitochondria in vitro have been investigated. Our results indicate that the inverse of the square of oscillatory period is linear with respect to (a) the permeant cation concentration; (b) the inverse of the permeant weak acid anion concentration, and (c) the rate of energyproduction. It has been shown that various factors contribute to damping of the oscillations. These factors include: substrate utilization, mitochondrial deterioration, imperfect mitochondrial synchronization, and, possibly, an oscillatory mechanism which dictates damping. An increased period length and extensive damping of oscillations occurs at a critical mitochondrial protein concentration (less than 0.6 mg protein/ml). Such inhibition can be reversed by the addition of cytochrome c.