The effect of medium tonicity on the rate of respiration and oxidative phosphorylation in liver mitochondria of active and hibernating ground squirrels

The rate of respiration and ATP synthesis in liver mitochondria (M) isolated from hibernating ground squirrels and incubated in the medium with normal tonicity (250 mosm) was shown to be considerably lower than the rate of respiration and ATP synthesis in liver M from active animals. The increase of the medium tonicity to 600 mosm simulated the state of M from hibernating animals, resulting in a decrease of the respiration rate of M from active ground squirrels. On the contrary, the decrease of the tonicity to 60 mosm caused the activation of the respiration and increase of the ATP synthesis in M from hibernating ground squirrels. Bromophenacylbromide (BPhB), an inhibitor of phospholipase A2, prevented the activation of the respiration of M from hibernating animals incubated in the medium with low tonicity. BPhB had practically no effect on the respiration of M from both hibernating and active ground squirrels as well as on the swelling of M in hypotonic medium. It was concluded that the activation of the respiration and increase of the ATP synthesis rate in M from hibernating ground squirrels incubated in the medium with low tonicity is related to the activation of phospholipase A2. It was assumed that decrease of phospholipase A2 activity and change in the lipid composition of mitochondrial membrane may be one of the reasons for inhibition of the respiration rate in M from hibernating ground squirrels.     

Phospholipase A activity determines the rate of respiration of the mitochondria in hibernating animals

The mechanisms for regulating the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels were studied. The microviscosity of the mitochondrial membrane in hibernating squirrels was found to be higher than that in active animals. Probably, a high microviscosity of the membrane causes a decreases in the rate of the transport of oxidation substrates into the mitochondrial matrix, which in turn may be one of the main reasons for the inhibition of mitochondrial respiration in hibernating squirrels. The activation of phospholipase A2 in a hypotonic medium results in the acceleration of the respiration and phosphorylation in the mitochondria from hibernating squirrels and is accompanied by the increase of the transport of substrates across the mitochondrial membrane. The inhibition of phospholipase A2 decreases Ca2+--induced acceleration of the transport of substrates and prevents the activation of the respiration and phosphorylation in a hypotonic medium.     

Biochemical bases of the inhibition and activation of liver mitochondrial respiration in hibernating susliks

The possible mechanisms in regulation of the respiration rate of mitochondria from liver of hibernating ground squirrels have been investigated. The inhibition of respiration has been shown to be mainly due to the inhibition of electron transfer to the respiratory chain from flavoproteins to cytochrome c. Calcium ions evoke the gradual increase in the respiration rate of mitochondria from liver of hibernating ground squirrels which is abolished by adding albumin, ruthenium red and sovcaine. The lower content of free fatty acids and decreased rate of the oxidation of exogenic NADH in the external pathway indicate the decrease in the activity of phospholipase A2 in mitochondria from liver of hibernating ground squirrels. The decreased calcium capacity of mitochondria indicates the higher sensitivity to calcium ions. A conclusion concerning the leading role of phospholipase A2 in Ca-induced activation of respiration of mitochondria from liver of hibernating ground squirrels is made.     

The inhibition of succinate, beta-oxybutyrate and glutamate transport in the liver mitochondria of hibernating susliks

Studies have been made on the permeability of the inner membrane of the liver mitochondria from hibernating and active ground squirrels for succinate, glutamate, hydroxybutyrate and inorganic phosphate. The permeability was calculated from the rate of mitochondrial swelling in 100 mM ammonium salts of the substrates and phosphate. It was shown that the rate of mitochondrial swelling in hibernating animals is 2--3 times lower than in active ones, being essentially identical in a solution of ammonium phosphate. It was concluded that the permeability of the inner mitochondrial membrane for the substrates decreases in hibernating animals, remaining unaffected for phosphate. Calcium-induced activation of membrane phospholipase A2 facilitates the transport of oxidative substrates into the mitochondria of hibernating ground squirrels, significant increase in the mitochondrial respiration being simultaneously observed. The data obtained suggest that inhibition of transport of oxidative substrates is one of the main factors which account for a low respiration rate in the mitochondria of hibernating animals.     

Carboxyatractylate-sensitive uncoupling in liver mitochondria from ground squirrels during hibernation and arousal

Energy coupling parameters of liver mitochondria from hibernating and arousing ground squirrels have been studied. In the oligomycin-treated mitochondria, carboxyatractylate, an inhibitor of the ATP/ADP-antiporter, is shown to decrease the respiration rate, to increase the membrane potential and to lower the rate of the membrane-potential discharge after the addition of cyanide to liver mitochondria from hibernating and arousing animals. BSA effectively substitutes for carboxyactactylate so that carboxyactactylate, added after BSA, has no effect. In mitochondria from hibernating animals, the maximal respiration rate in the presence of DNP and the rate of the membrane potential discharge in its absence are much lower than in those from arousing animals. It has been concluded that upon arousal of the animals from hibernation, the uncoupling of oxidative phosphorylation, mediated by free fatty acids and ATP/ADP-antiporter, parallels the respiratory chain activation.     

Inhibition of succinate oxidation and K+ transport in mitochondria during hibernation

Respiration of liver mitochondria of ground squirrels changes with physiological state. The inhibition of respiration at the level of dehydrogenases occurs during hibernation which is spontaneously removed during arousal. The main mechanism causing a decrease in respiration during hibernation seems to be the inhibition of succinate oxidation, induced by oxaloacetic acid. This is evidenced by the removal of the inhibition by glutamic and isocitric acids. A close correlation between the changes of K+ transport in mitochondria and of the physiological state of hibernator is observed. During hibernation the K+ transport rate decreases 3 times and during arousal it increases 1.5-fold in comparison with the active animals. The K+ content in mitochondria of hibernating and active ground squirrels is the same, whereas during arousal it increases 2-fold.     

The intensity of oxidative phosphorylation and the function of the adenylate system in the liver mitochondria of active and hibernating susliks Citellus undulatus

The state of adenylate system and intensity of oxidative phosphorylation in liver mitochondria of active and hibernating ground squirrels were studied depending on the concentration of extramitochondrial Ca2+ ([Ca2+]ex). It was shown that at [Ca2+]ex.10(-7) M, the content of ATP as well as ATP/ADP ratio are slightly lower in the mitochondria of hibernating ground squirrels than in the mitochondria of active animals. The other parameters of the adenylate system under the same conditions differ insignificantly. [Ca2+]ex increase to 10(-6) M has little effect on the parameters of the adenylate system of active animals. On the contrary, the mitochondria of hibernating ground squirrels are strongly affected: the level of ATP is 1.5-fold and the ratio of ATP/ADP is almost 2-fold decreased. At both [Ca2+]ex the intensity of oxidative phosphorylation is essentially higher in the mitochondria of active ground squirrels. With increasing [Ca2+]ex the rate of ATP synthesis decreases, and in the mitochondria of hibernating animals the decrease is more pronounced than in the mitochondria of active animals. Thus, oxidative phosphorylation and adenylate system of mitochondria from hibernating ground squirrels are more sensitive to [Ca2+]ex increase than those of the mitochondria of active animals.     

Mitochondrial metabolism in hibernation: metabolic suppression, temperature effects, and substrate preferences

We compared liver and skeletal muscle mitochondrial function among activity states to characterize regulated reversible metabolic suppression in the mammalian hibernator Spermophilus tridecemlineatus. At 37 degrees C, succinate oxidation was 70% lower in the liver mitochondria from torpid animals than in those from summer-active animals or in animals arousing from torpor. Respiration was very sensitive to temperature (Q(10) 5.8-9.8), and when measured at 25 degrees or 5 degrees C there was no difference among the three states. Liver mitochondria from summer-active animals oxidized pyruvate and beta -hydroxybutyrate at higher rates than those from torpid animals, and flux through complex 4 of the electron transport chain was about three- and fivefold higher than flux through complexes 2-4 and complexes 1-4, respectively. In the hibernating and arousing animals there was no difference in flux through complexes 2-4 and complex 4, suggesting a downregulation of cytochrome c oxidase in liver mitochondria during the hibernation season. Muscle mitochondrial respiration did not differ between the torpid and summer-active states in any of the parameters measured. The data support a regulated, reversible decrease of liver (but not muscle) mitochondrial oxidative phosphorylation in hibernating ground squirrels.     

Hepatic gluconeogenesis and mitochondrial function during hibernation

1. The aim of these studies was to investigate a mitochondrial basis for changes in gluconeogenesis during hibernation. 2. State 3 respiration rates in liver mitochondria from hibernating ground squirrels were reduced by 62-66%. The limiting reaction appeared to be electron transport, particularly in respiratory complex III. 3. The mitochondrial ATP + ADP + AMP content was reduced by 29% during hibernation; cellular adenine nucleotide content was unchanged. 4. Pyruvate carboxylation in intact mitochondria was decreased 75% during hibernation, although total pyruvate carboxylase activity was not lower. 5. Rates of gluconeogenesis in intact hepatocytes isolated from hibernators were lower than in cells from non-hibernators.     

Activation of oxidative phosphorylation and energy-dependent absorption of Ca2+ and K+ ions by liver mitochondria of hibernating ground squirrels in hypotonic media]

Activation of initially suppressed oxidative phosphorylation and energy-dependent uptake of Ca2+ and K+ ions by liver mitochondria of hibernating gophers which is prevented by phospholipase A2 inhibitors, has been shown to occur in hypotonic media. Partial inhibition of the respiratory chain of liver mitochondria of active gophers by antimycin A which causes a decrease in the uncoupled respiration rate and delta psi down to values typical of mitochondria of hibernating gophers, practically exactly reproduced the suppression of oxidative phosphorylation and energy-dependent uptake of cations observed during hibernation. It was concluded that partial deenergization arising as a result of inhibition of the respiratory chain is the main and unique cause of suppression of energy-dependent functions of liver mitochondria of hibernating gophers.     

The mechanism of calcium-dependent activation of respiration of liver mitochondria from hibernating ground squirrels,Citellus undulatus

• 1.1. It is shown that Ca²⁺-dependent activation of respiration of liver mitochondria from hibernating ground squirrels is accompanied by mitochondrial swelling.
• 2.2. The swelling of mitochondria from hibernating ground squirrels, as well as the activation of mitochondrial respiration, is precluded by cyclosporin A, p-bromphenacylbromide and oligomycin. Carboxyatractiloside, on the contrary, under these conditions favors the swelling and the acceleration of respiration.
• 3.3. It was concluded that Ca²⁺-dependent activation of hibernating ground squirrel liver mitochondrial respiration resulted from the appearance of a non-specific permeability pathway and from swelling of mitochondria.

     

Tissue-specific depression of mitochondrial proton leak and substrate oxidation in hibernating arctic ground squirrels

A significant proportion of standard metabolic rate is devoted to driving mitochondrial proton leak, and this futile cycle may be a site of metabolic control during hibernation. To determine if the proton leak pathway is decreased during metabolic depression related to hibernation, mitochondria were isolated from liver and skeletal muscle of nonhibernating (active) and hibernating arctic ground squirrels (Spermophilus parryii). At an assay temperature of 37 degrees C, state 3 and state 4 respiration rates and state 4 membrane potential were significantly depressed in liver mitochondria isolated from hibernators. In contrast, state 3 and state 4 respiration rates and membrane potentials were unchanged during hibernation in skeletal muscle mitochondria. The decrease in oxygen consumption of liver mitochondria was achieved by reduced activity of the set of reactions generating the proton gradient but not by a lowered proton permeability. These results suggest that mitochondrial proton conductance is unchanged during hibernation and that the reduced metabolism in hibernators is a partial consequence of tissue-specific depression of substrate oxidation.     

Analysis of the causes of the suppression of oxidative phosphorylation and energy-dependent cationic transport into liver mitochondria of hibernating gophers, Citellus undulatus.

1. The causes of the suppression of oxidative phosphorylation and energy-dependent cationic transport into liver mitochondria of hibernating gophers have been analysed. 2. The decrease of the ATP synthesis rate and suppression of the energy-dependent K(+)- and Ca(2+)-transport into mitochondria during hibernation has been found to be mainly related to a delta psi decrease in mitochondria of hibernating gophers. 3. The increase delta psi upon incubation of the mitochondria of hibernating animals in a hypotonic medium results in an essential acceleration of ATP synthesis and energy-dependent cationic transport.     

Participation of the ATP/ADP antiporter and fatty acids in oxidative phosphorylation uncoupling in squirrel liver mitochondria during winter hibernation and awakening]

The parameters of energy coupling of mitochondria isolated from the livers of hibernating and awakening gophers were studied. The ATP/ADP-antiporter inhibitor carboxyatractylate slowed down the respiration rate, increased delta psi and decreased the ionic conductivity of the inner mitochondrial membrane as measured by the rate of the delta psi decline after addition of cyanide (in the presence of oligomycin and EGTA). A similar effect was produced by BSA, carboxyatractylate being fairly ineffective in the presence of BSA. In hibernating gophers the maximal rate of the uncoupled respiration and the ionic conductivity of the inner mitochondrial membrane were markedly decreased as compared with awakening gophers. The data obtained suggest that in awakening animals fatty acids induce the uncoupling of oxidative phosphorylation by the ATP/ADP-antiporter, this process being simultaneous with the activation of the respiratory chain.     

Control of mitochondrial respiration by the phosphate potential

The rate of respiration of suspensions of mitochondria in the presence of excess oxygen and substrate is shown to be dependent on the ratio of the concentration of adenosine triphosphate (ATP) to the product of the concentrations of adenosine diphosphate and orthophosphate. The mitochondrial respiratory chain is essentially in equilibrium with the reactions for ATP synthesis. The rate of mitochondrial respiration is controlled by the free energy requirement for ATP synthesis and this control is expressed on the rates of the reactions for reduction of the dehydrogenases by substrate and the oxidation of cytochrome $\text{a}{}_3$ by molecular oxygen.     

The effect of hibernation on lipids of the liver mitochondrial fraction in the Yakut ground squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

We demonstrated that the level of phospholipids in the liver mitochondrial fraction is increased by 60% during the winter hibernation season in the Yakut ground squirrel S. undulatus; the phospholipid composition in sleeping animals is characterized by an increase in phosphatidylethanolamine compared with summer animals. A sharp increase in the level of cholesterol, as well as fatty acid, monoglycerides, and diglycerides was found in the mitochondrial fraction of hibernating ground squirrels in relation to summer ground squirrels. Functional changes during hibernation concern the level of phosphatidylserine (the growth in sleeping animals compared with active animals). Seasonal modification of the lipid composition of the liver mitochondria (particularly, an increase in the level of cholesterol) can play a role in the resistance of mitochondria to the seasonal increase in the level of fatty acids in the liver. Lipids of the liver mitochondrial fraction are involved in the ground squirrel adaptation to the hibernation season.     

Mitochondrial metabolic suppression and reactive oxygen species production in liver and skeletal muscle of hibernating thirteen-lined ground squirrels

During hibernation, animals cycle between periods of torpor, during which body temperature (T(b)) and metabolic rate (MR) are suppressed for days, and interbout euthermia (IBE), during which T(b) and MR return to resting levels for several hours. In this study, we measured respiration rates, membrane potentials, and reactive oxygen species (ROS) production of liver and skeletal muscle mitochondria isolated from ground squirrels (Ictidomys tridecemlineatus) during torpor and IBE to determine how mitochondrial metabolism is suppressed during torpor and how this suppression affects oxidative stress. In liver and skeletal muscle, state 3 respiration measured at 37°C with succinate was 70% and 30% lower, respectively, during torpor. In liver, this suppression was achieved largely via inhibition of substrate oxidation, likely at succinate dehydrogenase. In both tissues, respiration by torpid mitochondria further declined up to 88% when mitochondria were cooled to 10°C, close to torpid T(b). In liver, this passive thermal effect on respiration rate reflected reduced activity of all components of oxidative phosphorylation (substrate oxidation, phosphorylation, and proton leak). With glutamate + malate and succinate, mitochondrial free radical leak (FRL; proportion of electrons leading to ROS production) was higher in torpor than IBE, but only in liver. With succinate, higher FRL likely resulted from increased reduction state of complex III during torpor. With glutamate + malate, higher FRL resulted from active suppression of complex I ROS production during IBE, which may limit ROS production during arousal. In both tissues, ROS production and FRL declined with temperature, suggesting ROS production is also reduced during torpor by passive thermal effects.     

Involvement of the energy-dissipating systems in modulating the energetic efficiency of respiration in mitochondria from etiolated winter wheat seedlings

Effects of cyanide-resistant alternative oxidase (AOX) and modulators of plant uncoupling mitochondrial proteins (PUMP) on respiration rate and generation of transmembrane electric potential (ΔΨ) were investigated during oxidation of various substrates by isolated mitochondria from etiolated coleoptiles of winter wheat (Triticum aestivum L.). Oxidative phosphorylation in wheat mitochondria during malate and succinate oxidation was quite effective (it was characterized by high respiratory control ratio as defined by Chance, high ADP/O ratio, and rapid ATP synthesis). Nevertheless, the effectiveness of oxidative phosphorylation was substantially modulated by operation of energy-dissipating systems. The application of safranin dye revealed the partial dissipation of ΔΨ during inhibition of cytochrome-mediated malate oxidation by cyanide and antimycin A and demonstrated the operation of AOX-dependent compensatory mechanism for ΔΨ generation. The complex I of mitochondrial electron transport chain was shown to play the dominant role in ΔΨ generation and ATP synthesis during AOX functioning upon inhibition of electron transport through the cytochrome pathway. Effects of linoleic acid (PUMP activator) at physiologically low concentrations (4–10 μM) on respiration and ΔΨ generation in mitochondria were examined. The uncoupling effect of linoleic acid was shown in activation of the State 4 respiration, as well as in ΔΨ dissipation; this effect was eliminated in the presence of BSA but was insensitive to purine nucleotides. The uncoupling effect of linoleic acid was accompanied by reversible inhibition of AOX activity. The results are discussed with regard to possible physiological role of mitochondrial energy-dissipating systems in regulation of energy transduction in plant cells under stress conditions.     

Effects of warm and cold ischemia on mitochondrial functions in brain,liver and kidney

The purpose of this work was to study the effects of warm (37°C) and cold (4°C) ischemia on different mitochondrial functions in rat brain, liver and kidney.After l0 to 60 minutes of ischemia at 37°C the energy coupled respiration as well as the ADP-induced malate-aspartate shuttle activity in brain and liver mitochondria or the rate of mitochondrial ATP synthesis in kidney were significantly decreased. However, the respiratory rates and the shuttle activity in the absence of ADP remained unchanged. These data suggest that ischemia primarily affects electron transport in the respiratory chain rather than the hydrogen shuttle and the energy coupling system. When the temperature during the indicated ischemic periods was decreased to 4°C, in brain and liver no significant alterations of these mitochondrial functions were found in comparison with the non-ischemic controls. When rat kidneys were stored for 36 hours at 4°C according to Collins mimicing transplantation conditions, the mitochondrial respiration and ATP synthesis were only slightly decreased. It therefore appears that hypothermia can prevent effectively mitochondrial dysfunction due to ischemia.     

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.