Long-chain 3-hydroxy fatty acids accumulating in long-chain 3-hydroxyacyl-CoA dehydrogenase and mitochondrial trifunctional protein deficiencies uncouple oxidative phosphorylation in heart mitochondria

Cardiomyopathy is a common clinical feature of some inherited disorders of mitochondrial fatty acid β-oxidation including mitochondrial trifunctional protein (MTP) and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies. Since individuals affected by these disorders present tissue accumulation of various fatty acids, including long-chain 3-hydroxy fatty acids, in the present study we investigated the effect of 3-hydroxydecanoic (3 HDCA), 3-hydroxydodecanoic (3 HDDA), 3-hydroxytetradecanoic (3 HTA) and 3-hydroxypalmitic (3 HPA) acids on mitochondrial oxidative metabolism, estimated by oximetry, NAD(P)H content, hydrogen peroxide production, membrane potential (ΔΨ) and swelling in rat heart mitochondrial preparations. We observed that 3 HTA and 3 HPA increased resting respiration and diminished the respiratory control and ADP/O ratios using glutamate/malate or succinate as substrates. Furthermore, 3 HDDA, 3 HTA and 3 HPA decreased ΔΨ, the matrix NAD(P)H pool and hydrogen peroxide production. These data indicate that these fatty acids behave as uncouplers of oxidative phosphorylation. We also verified that 3 HTA-induced uncoupling-effect was not mediated by the adenine nucleotide translocator and that this fatty acid induced the mitochondrial permeability transition pore opening in calcium-loaded organelles since cyclosporin A prevented the reduction of mitochondrial ΔΨ and swelling provoked by 3 HTA. The present data indicate that major 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies behave as strong uncouplers of oxidative phosphorylation potentially impairing heart energy homeostasis.     

Mitochondrial bioenergetics deregulation caused by long-chain 3-hydroxy fatty acids accumulating in LCHAD and MTP deficiencies in rat brain: A possible role of mPTP opening as a pathomechanism in these disorders?

Long-chain 3-hydroxylated fatty acids (LCHFA) accumulate in long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies. Affected patients usually present severe neonatal symptoms involving cardiac and hepatic functions, although long-term neurological abnormalities are also commonly observed. Since the underlying mechanisms of brain damage are practically unknown and have not been properly investigated, we studied the effects of LCHFA on important parameters of mitochondrial homeostasis in isolated mitochondria from cerebral cortex of developing rats. 3-Hydroxytetradecanoic acid (3 HTA) reduced mitochondrial membrane potential, NAD(P)H levels, Ca^2 + retention capacity and ATP content, besides inducing swelling, cytochrome c release and H₂O₂ production in Ca^2 +-loaded mitochondrial preparations_. We also found that cyclosporine A plus ADP, as well as ruthenium red, a Ca^2 + uptake blocker, prevented these effects, suggesting the involvement of the mitochondrial permeability transition pore (mPTP) and an important role for Ca^2 +, respectively. 3-Hydroxydodecanoic and 3-hydroxypalmitic acids, that also accumulate in LCHAD and MTP deficiencies, similarly induced mitochondrial swelling and decreased ATP content, but to a variable degree pending on the size of their carbon chain. It is proposed that mPTP opening induced by LCHFA disrupts brain bioenergetics and may contribute at least partly to explain the neurologic dysfunction observed in patients affected by LCHAD and MTP deficiencies.     

OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA OF DEVELOPING RAT BRAIN

Abstract—

• 1 Oxygen uptake, ADP/O ratios and respiratory control ratios (RCR) were studied by oxygen electrode techniques in mitochondria prepared from developing rat brain.
• 2 Oxygen consumption, ADP/O ratios and RCR based on mitochondrial protein concentration increased with maturation. Of the substrates employed, succinate supported oxygen uptake best and malate poorest.
• 3 The addition of exogenous NAD to the mitochondrial preparation had no effect on rate of oxygen uptake.
• 4 Lack of change in ADP/O ratio in the presence of glucose, a tricarboxylic acid cycle intermediate (α-oxoglutarate), and ATP leads us to believe that there is no significant hexokinase activity in this preparation.

     

Rotenone-like action of the branched-chain phytanic acid induces oxidative stress in mitochondria

Phytanic acid (Phyt) increase is associated with the hereditary neurodegenerative Refsum disease. To elucidate the still unclear toxicity of Phyt, mitochondria from brain and heart of adult rats were exposed to free Phyt. Phyt at low micromolar concentrations (maximally: 100 nmol/mg of protein) enhances superoxide (O(2)(.))(2) generation. Phyt induces O(2)(.) in state 3 (phosphorylating), as well as in state 4 (resting). Phyt stimulates O(2)(.) generation when the respiratory chain is fed with electrons derived from oxidation of glutamate/malate, pyruvate/malate, or succinate in the presence of rotenone. With succinate alone, Phyt suppresses O(2)(.) generation caused by reverse electron transport from succinate to complex I. The enhanced O(2)(.) generation by Phyt in state 4 is in contrast to the mild uncoupling concept. In this concept uncoupling by nonesterified fatty acids should abolish O(2)(.) generation. Stimulation of O(2)(.) generation by Phyt is paralleled by inhibition of the electron transport within the respiratory chain or electron leakage from the respiratory chain. The interference of Phyt with the electron transport was demonstrated by inhibition of state 3- and p-trifluoromethoxyphenylhydrazone (FCCP)-dependent respiration, inactivation of the NADH-ubiquinone oxidoreductase complex in permeabilized mitochondria, decrease in reduction of the synthetic electron acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in state 4, and increase of the mitochondrial NAD(P)H level in FCCP-uncoupled mitochondria. Thus, we suggest that complex I is the main site of Phyt-stimulated O(2)(.) generation. Furthermore, inactivation of aconitase and oxidation of the mitochondrial glutathione pool show that enhanced O(2)(.) generation with chronic exposure to Phyt causes oxidative damage.     

Suppression of the mitochondrial oxidation of (-)-palmitylcarnitine by the malate-aspartate and alpha-glycerophosphate shuttles.

Palmitylcarnitine oxidation by isolated liver mitochondria has been used to investigate the interaction of fatty acid oxidation with malate, glutamate, succinate, and the malate-aspartate shuttle. Mitochondria preincubated with fluorocitrate were added to a medium containing 2mM ATP and ATPase. This system, characterized by a high energy change, allowed titration of respiration to any desired rate between States 4 and 3 (Chance, B., and Williams, G. R. (1956) Adv. Enzymol. Relat. Areas Mol. Biol. 17, 65-134). When respiration (reference, with palmitylcarnitine and malate as substrates) was set at 75% of State 3, the oxidation of palmitylcarnitine was limited by acetoacetate formation. The addition of malate or glutamate approximately doubled the rate of beta oxidation. Malate circumvented this limitation by citrate formation, but the effect of glutamate apparently was due to enhancement of the capacity for ketogenesis. The rate of beta oxidation was curtailed when malate and glutamate were both present. This curtailment was more pronounced when the malate-aspartate shuttle was fully reconstituted. Among the oxidizable substrates examined, succinate was most effective in inhibiting palmitylcarnitine oxidation. Mitochondrial NADH/NAD+ ratios were correlated positively with suppression of beta oxidation. The degree of suppression of beta oxidation by the malate-aspartate shuttle (NADH oxidation) or by succinate oxidation was dependent on the respiratory state. Both substrates extensively reduced mitochondrial NAD+ and markedly suppressed beta oxidation as respiration approached State 4. Calculations of the rates of flux of hydrogen equivalents through beta oxidation show that the suppression of beta oxidation by glutamate or by the malate-aspartate shuttle is accounted for by increased flux of reducing equivalents through mitochondrial malic dehydrogenase. This increased Flux is accompanied by an increase in the steady state NADH/NAD+ ratio and a marked decrease in the synthesis of citrate. The alpha-glycerophosphate shuttle was reconstituted with mitochondria isolated from rats treated with L-thyroxine. This shuttle was about equal to the reconstructed malate-aspartate shuttle in supression of palmitylcarnitine oxidation. This interaction could not be demonstrated in euthyroid animals owing to the low activity of the mitochondrial alpha-glycerol phosphate dehydrogenase. It is concluded that beta oxidation can be regulated by the NADH/NAD+ ratio. The observed stimulation of flux through malate dehydrogenase both by glutamate and by the malate-aspartate shuttle results in an increased steady state NADH/NAD+ ratio, and is linked to a stoichiometric outward transport of aspartate. We suggest, therefore, that some of the reducing pressure exerted by the malate-aspartate shuttle and by glutamate plus malate is provided through the energy-linked, electrogenic transport of aspartate out of the mitochondria. These results are discussed with respect to the mechanism of the genesis of ethanol-induced fatty liver.     

Action of diclofenac on kidney mitochondria and cells

The mitochondrial membrane potential measured in isolated rat kidney mitochondria and in digitonin-permeabilized MDCK type II cells pre-energized with succinate, glutamate, and/or malate was reduced by micromolar diclofenac dose-dependently. However, ATP biosynthesis from glutamate/malate was significantly more compromised compared to that from succinate. Inhibition of the malate-aspartate shuttle by diclofenac with a resultant decrease in the ability of mitochondria to generate NAD(P)H was demonstrated. Diclofenac however had no effect on the activities of NADH dehydrogenase, glutamate dehydrogenase, and malate dehydrogenase. In conclusion, decreased NAD(P)H production due to an inhibition of the entry of malate and glutamate via the malate-aspartate shuttle explained the more pronounced decreased rate of ATP biosynthesis from glutamate and malate by diclofenac. This drug, therefore affects the bioavailability of two major respiratory complex I substrates which would normally contribute substantially to supplying the reducing equivalents for mitochondrial electron transport for generation of ATP in the renal cell.     

Regulation of the mitochondrial oxidation-reduction state in intact hepatocytes by calcium ions

The mitochondrial NADH:NAD ratio of isolated intact liver cells incubated in calcium-free Hanks solution, in the endogenous state or with lactate, alanine, α-ketoglutarate, glutamate, fumarate, malate, or albumin-bound palmitate, was elevated by 1 mM CaCl₂. The chloride salts of Ba⁺², Cd⁺², Cu⁺², Mn⁺², Sr⁺², Zn⁺², Al⁺³, Ce⁺³ and La⁺³ caused no such change. In contrast, calcium decreased the mitochondrial NADH:NAD ratio of hepatocytes incubated with succinate. Calcium did not affect the NADH:NAD ratio in the liver cell cytosol or the energy charge. The calcium-induced elevation in the mitochondrial NADH:NAD ratio was reversed by the uncoupler 1799. These observations demonstrate a specific effect of calcium ions in the regulation of the mitochondrial oxidation-reduction state in intact liver cells.     

Respiratory Properties of Mitochondria from Rice Seedlings Germinated under Water and Their Changes during Air Adaptation

Respiratory activities were compared among rice seedlings germinated in air for 6 days (aerobic seedlings), those germinated under water for 5 days (submerged seedlings), and those grown in air for 1 day after 5 days' submerged germination (air-adapted seedlings). The respiratory activity of the submerged seedlings increased rapidly on transfer to air and reached a plateau at 16 hours in air. Respiration of the submerged seedlings was as sensitive to cyanide as those of aerobic and air-adapted seedlings. 2,4-Dinitrophenol had no effect on the respiration of the submerged seedlings, but stimulated those of the other two types of seedlings. Mitochondria from three types of seedlings did not differ in the ADP/O ratio and the respiratory control ratio (RCR) when succinate was oxidized. However, mitochondria from submerged seedlings (submerged mitochondria) showed poor RCR of about unity when malate was oxidized. Both the rate of succinate oxidation and succinate dehydrogenase activity were low in submerged mitochondria, but increased during air adaptation. Although submerged mitochondria oxidized malate very slowly, this activity increased after exposure to air without any increase in malate dehydrogenase activity. When NAD⁺ was added to submerged mitochondria, oxidation of malate was restored to the level of the aerobic controls. Addition of NAD⁺ enhanced the state 3 rate in submerged mitochondria, and RCR recovered to nearly the same value as that of the aerobic controls. Similar effects of NAD⁺ on 2-oxoglutarate oxidation were observed. All these defects in submerged mitochondria were repaired during air adaptation. These results suggest that NAD⁺-linked substrate oxidation was low in submerged mitochondria because of NAD⁺ deficiency, and that the oxidation increased with an increasing level of NAD⁺ during air adaptation.     

Age-associated deficit of mitochondrial oxidative phosphorylation in skeletal muscle: Role of carnitine and lipoic acid

Mitochondrial damage has implicated a major contributor for ageing process. In the present study, we measured mitochondrial membrane swelling, mitochondrial respiration (state 3 and 4) by using oxygen electrode in skeletal muscle of young (3–4 months old) and aged rats (above 24 months old) with supplementation of l-carnitine and dl-α-lipoic acid. Our results shows that the mitochondrial membrane swelling and state 4 respiration were increased more in skeletal muscle mitochondria of aged rats than in young control rats, whereas the state 3 respiration, respiratory control ratio (RCR) and ADP:O ratio decreased more in aged rats than in young rats. After supplementation of carnitine and lipoic acid to aged rats for 30 days, the state 3 respiration and RCR were increased, whereas the state 4 and mitochondrial membrane swelling were decreased to near normal rats. From our results, we conclude that combined supplementation of carnitine and lipoic acids to aged rats increases the skeletal muscle mitochondrial respiration, thereby increasing the level of ATP. (Mol Cell Biochem xxx: 83–89, 2005)     

High vulnerability of the heart and liver to 3‐hydroxypalmitic acid–induced disruption of mitochondrial functions in intact cell systems

Patients affected by long‐chain 3‐hydroxyacyl‐CoA dehydrogenase (LCHAD) deficiency predominantly present severe liver and cardiac dysfunction, as well as neurological symptoms during metabolic crises, whose pathogenesis is still poorly known. In this study, we demonstrate for the first time that pathological concentrations of 3‐hydroxypalmitic acid (3HPA), the long‐chain hydroxyl fatty acid (LCHFA) that most accumulates in LCHAD deficiency, significantly decreased adenosine triphosphate‐linked and uncoupled mitochondrial respiration in intact cell systems consisting of heart fibers, cardiomyocytes, and hepatocytes, but less intense in diced forebrain. 3HPA also significantly reduced mitochondrial Ca²⁺ retention capacity and membrane potential in Ca²⁺‐loaded mitochondria more markedly in the heart and the liver, with mild or no effects in the brain, supporting a higher susceptibility of the heart and the liver to the toxic effects of this fatty acid. It is postulated that disruption of mitochondrial energy and Ca²⁺ homeostasis caused by the accumulation of LCHFA may contribute toward the severe cardiac and hepatic clinical manifestations observed in the affected patients.     

The Respiratory Chain of Plant Mitochondria: XI. Electron Transport from Succinate to Endogenous Pyridine Nucleotide in Mung Bean Mitochondria

Energy-linked reverse electron transport from succinate to endogenous NAD in tightly coupled mung bean (Phaseolus aureus) mitochondria may be driven by ATP if the two terminal oxidases of these mitochondria are inhibited, or may be driven by the free energy of succinate oxidation. This reaction is specific to the first site of energy conservation of the respiratory chain; it does not occur in the presence of uncoupler. If mung bean mitochondria become anaerobic during oxidation of succinate, their endogenous NAD becomes reduced in the presence of uncoupler, provided that both inorganic phosphate (P_i) and ATP are present. No reduction occurs in the absence of P_i, even in the presence of ATP added to provide a high phosphate potential. If fluorooxaloacetate is present in the uncoupled, aerobic steady state, no reduction of endogenous NAD occurs on anaerobiosis; this compound is an inhibitor of malate dehydrogenase. This result implies that endogenous NAD is reduced by malate formed from the fumarate generated during succinate oxidation. The source of free energy is most probably the endogenous energy stores in the form of acetyl CoA, or intermediates convertible to acetyl CoA, which removes the oxaloacetate formed from malate, thus driving the reaction towards reduction of NAD.     

Developmental Changes in the NAD Content in Sugar-Beet Root Mitochondria and Their Effect on the Oxidative Activity of These Organelles

The NAD content was determined in mitochondria isolated from sugar-beet roots at various stages of plant development. A high NAD content (7.6 ± 0.9 nmol/mg mitochondrial protein) was observed in the mitochondria of actively growing roots of 80–95-day-old plants, but it decreased ca. twofold by the end of the first year of plant development, before the roots were harvested for storage. The mitochondria isolated from roots stored at low temperature for two to three months and those after five to eight days of regrowth during the second year of plant development manifested an even lower NAD content (2.2 ± 0.4 and 2.0 ± 0.5 nmol/mg protein, respectively). A drastic decrease in the NAD content in mitochondria from stored roots did not result from the impairment of the inner membrane of these organelles and was evidently regulatory in its nature. The effect of developmental changes in the intramitochondrial NAD content on the malate oxidation pattern was studied. In the mitochondria of stored roots, the low NAD content limited the rate of malate oxidation in state 3, because the addition to the reaction mixture of exogenous NAD, which can be transported to the mitochondrial matrix, promoted malate oxidation by 30–50%. Rotenone inhibited malate oxidation in the stored-root mitochondria by more than 70%; in this case, the rate of rotenone-resistant malate oxidation in these organelles increased by several times in the presence of exogenous NAD. In the mitochondria of the growing root, exo-genous NAD did not affect the rate of malate oxidation, and rotenone inhibited it only by 25–35%. The analysis of the data obtained here and the published evidence suggests the existence of a universal mechanism of respiration control and the regulation of the functional activity of plant mitochondria. This mechanism acts through a change in the NAD content in the organelle matrix. This NAD can be used in the course of plant development, e.g., during the transition of sugar-beet-root cells in the dormant state, when the respiration rate must decline.     

Inhibition of anion transport across the mitochondrial membrane by amytal

It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K_i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p-trifluoromethoxyphenylhydrazone.Using [¹⁴C] succinate and [¹⁴C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P_i, succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P_i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [¹⁴C]succinate/anion (P_i, succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P_i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P_i translocation across the mitochondrial membrane.     

Comparative effects of three 1,4-dihydropyridine derivatives [OSI-1210, OSI-1211 (etaftoron), and OSI-3802] on rat liver mitochondrial bioenergetics and on the physical properties of membrane lipid bilayers: relevance to the length of the alkoxyl chain in positions 3 and 5 of the DHP ring

The 1,4-dihydropyridines OSI-1210, OSI-1211 (etaftoron), and OSI-3802 are compounds with similar chemical structure. They differ by the length of the alkoxyl chain in positions 3 and 5 of the dihydropyridine (DHP) ring and by their pharmacological action characteristics. However, as far as we know, a clear relationship between the effects of these compounds and the length of the alkoxyl chain in positions 3 and 5 of the DHP has not been established. The goal of this study was to compare the influence of OSI-1210, OSI-1211 (etaftoron), and OSI-3802 on rat liver mitochondrial bioenergetics and on the physical properties of membrane lipid bilayers, correlating their actions with the length of the alkoxyl chain in positions 3 and 5 of the DHP ring. Using either glutamate/malate or succinate as respiratory substrates, all the compounds, in concentrations of up to 500 microM, depressed state 3 and uncoupled respiration, respiratory control (RCR) and ADP/O ratios, and phosphorylation rate, whereas state 4 respiration was stimulated. However, the stimulatory effect on state 4 induced by OSI-3802, the compound with the longest chain in positions 3 and 5 of the DHP ring, as well as its inhibitory effects on RCR and ADP/O ratios and phosphorylation rate were more pronounced than that induced by OSI-1210 and OSI-1211 (etaftoron), the compounds with the shortest and intermediate chains, respectively. Moreover, OSI-3802 maximized state 4 stimulation and minimized RCR and ADP/O ratios, and phosphorylation rate at a concentration of 100 microM, whereas low graduate effects were detected with OSI-1210 and OSI-1211 (etaftoron) for concentrations of up to 500 microM. At low concentrations (< or =30 microM), OSI-3802, like its analogue OSI-1212 (cerebrocrast), reduced the phase transition temperature, the cooperative unit size, and the enthalpy associated with the phase transition temperature of dimyristoylphosphatidylcholine (DMPC) membrane bilayers. A good correlation was established between the effects of 200 microM OSI-1210, OSI-1211 (etaftoron), and OSI-3802 on glutamate/malate- and succinate-dependent RCR of rat liver mitochondria and on the enthalpy change (Delta H) for the thermotropic profile of DMPC membrane bilayers at a 0.2 drug/DMPC molar ratio, indicating that the changes induced by these compounds on both mitochondrial membrane integrity and physical properties of DMPC membrane bilayers are strongly related to the length of the alkoxyl chain in positions 3 and 5 of the DHP ring. A putative relationship between membrane physical perturbation, bioenergetics impairment and the molecular characteristics of the compounds will be established as an approach to better understand their differentiated toxicological and pharmacological actions.     

Effects of piperine on enzyme activities and bioenergetic functions in isolated rat liver mitochondria and hepatocytes

The influence of piperine on the enzymes and bioenergetic functions in isolated rat liver mitochondria and hepatocytes was studied. Piperine at lower concentrations (<50 μM) did not affect the RCR and ADP:O ratios, state 4 and 3 respirations supported by site-specific substrates, viz. glutamate + malate, succinate, and ascorbate + TMPD. The site-specific effects became significantly apparent only at higher concentrations. Only the state 3 respiration supported by NAD-linked substrates was impaired equipotently in mitochondria and permeabilized hepatocytes; the effect appeared to be localized at energy-coupling site 1. In hypotonic treated mitochondria, respiration supported by three kinds of substrates was not affected. Among the respiratory chain-linked enzymes, the activity of NADH-dehydrogenase registered a significant decrease of about 25, 42, and 53% at 100, 150, and 180 μM piperine, respectively. The activity of Mg⁺⁺-ATPase, however, was stimulated at concentrations above 150 μM. Among the matrix enzymes, only malate and succinate dehydrogen-ases were studied. Malate dehydrogenase only showed a strong concentration-related inhibition in both the forward and backward directions. Enzyme kinetics indicated noncompetitive inhibition with a very low Ki of 10 μM. The presence of unsaturated double bonds in the side chain of piperine appeared essential for producing this strong inhibition. The studies suggested that piperine produces concentration related site-specific effects on mitochondrial bioenergetics and enzymes of energy metabolism.     

Methoxyacetic acid and ethoxyacetic acid inhibit mitochondrial function in vitro

Ethylene glycol monomethyl ether (EGME) and ethylene glycol monoethyl ether (EGEE) have recently been shown to be potent reproductive toxicants in laboratory animals. The toxicity of these compounds is believed to be due to their metabolites, methoxyacetic acid (MAA) and ethoxyacetic acid (EAA). Since the primary targets of EGME and EGEE appear to be tissues with rapidly dividing cell systems and high rates of respiration and energy metabolism, the effects of these compounds and their proposed metabolites on mitochondria were investigated. At concentrations beginning at 3.85 mM, MAA and EAA inhibited state 3 respiration and the respiratory control ratio (RCR) in hepatic mitochondria with either succinate or citrate/malate as substrates. Cytochrome c oxidase activity was also inhibited by both metabolites at similar concentrations. The effects of MAA, the metabolite from the more potent compound, on testicular mitochondria were found to be comparable. Neither EGME or EGEE appeared to affect mitochondrial function at concentrations as high as 238 or 113 mM, respectively. These results support the hypothesis that the toxicity of EGME and EGEE are due to their metabolites, MAA and EAA, and that these metabolites may exert their effects, in part, on mitochondrial function.     

Oxaloacetate and malate transport by plant mitochondria

The permeability of mitochondria from pea (Pisum sativum L. var Kleine Rheinländerin) leaves, etiolated pea shoots, and potato (Solanum tuberosum) tuber for malate, oxaloacetate, and other dicarboxylates was investigated by measurement of mitochondrial swelling in isoosmolar solutions of the above mentioned metabolites. For the sake of comparison, parallel experiments were also performed with rat liver mitochondria. Unlike the mammalian mitochondria, the plant mitochondria showed only little swelling in ammonium malate plus phosphate media but a dramatic increase of swelling on the addition of valinomycin. Similar results were obtained with oxaloacetate, maleate, fumarate, succinate, and malonate. n-Butylmalonate and phenylsuccinate, impermeant inhibitors of malate transport in mammalian mitochondria, had no marked inhibitory effect on valinomycin-dependent malate and oxaloacetate uptake of the plant mitochondria. The swelling of plant mitochondria in malate plus valinomycin was strongly inhibited by oxaloacetate, at a concentration ratio of oxaloacetate/malate of 10⁻³. From these findings it is concluded: (a) In a malate-oxaloacetate shuttle transferring redox equivalents from the mitochondrial matrix to the cytosol, malate and oxaloacetate are each transported by electrogenic uniport, probably linked to each other for the sake of charge compensation. (b) The transport of malate between the mitochondrial matrix and the cytosol is controlled by the oxaloacetate level in such a way that a redox gradient can be maintained between the NADH/NAD systems in the matrix and the cytosol. (c) The malate-oxaloacetate shuttle functions mainly in the export of malate from the mitochondria, whereas the import of malate as a respiratory substrate may proceed by the classical malate-phosphate antiport.     

The effect of acylcarnitines on the oxidation of branched chain alpha-keto acids in mitochondria

The oxidation of 14C-labelled branched-chain alpha-keto acids corresponding to the branched-chain amino acids valine, isoleucine and leucine has been studied in isolated mitochondria from heart, liver and skeletal muscle. 1. Heart and liver mitochondria have similar capacities to oxidize these alpha-keto acids based on protein content. Skeletal muscle mitochondria also show significant activity. 2. Half maximum rates are obtained with approximately 0.1 mM of the alpha-keto acids under optimal conditions. Added NAD and CoA had no effect on the oxidation rate, showing that endogenous mitochondrial NAD and CoA are required for the oxidation. 3. Addition of carnitine esters of fatty acids (C6--C16), succinate, pyruvate, or alpha-ketoglutarate inhibited the oxidation of the branched chain alpha-keto acids, especially in a high-energy state (no ADP added). In heart mitochondria the addition of AD (low-energy state) decreased the inhibitory effects of acylcarnitines of medium chain length or of pyruvate, and abolished the inhibitory effect of succinate. It is suggested that the oxidation rate is regulated mainly by the redox state of the mitochondria under the conditions used. 4. The results are discussed in relation to the regulation of branched-chain amino acid metabolism in the body.     

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), cyperquat (MPP+) and paraquat on isolated mitochondria from rat striatum, cortex and liver

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), its metabolite 1-methyl-4-phenyl pyridinium ion (MPP+, cyperquat) and a structurally-related compound paraquat on mitochondrial functions were investigated in isolated organelles from rat striatum, cortex and liver. MPTP (0.1-1.0 mM) had no significant effect on various parameters of mitochondrial oxidative phosphorylation. In contrast, MPP+ (0.5 mM) inhibited the oxidation of the nicotinamide adenine dinucleotide (NAD+)-linked substrates pyruvate and malate but not that of the flavin adenine dinucleotide (FAD+)-linked substrate succinate. Paraquat (5.0 mM) significantly stimulated basal oxygen consumption (state 4) without influencing the oxygen utilization (state 3) associated with adenosine diphosphate (ADP) phosphorylation. Thus, these structurally-related compounds have different effects on mitochondrial oxidative phosphorylation, but the organelles from striatum, cortex and liver were affected in a similar manner by these compounds.