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

1. It is shown that Ca(2+)-dependent activation of respiration of liver mitochondria from hibernating ground squirrels is accompanied by mitochondrial swelling. 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. It was concluded that Ca(2+)-dependent activation of hibernating ground squirrel liver mitochondrial respiration resulted from the appearance of a non-specific permeability pathway and from swelling of mitochondria.

Relationship between structure and function of liver mitochondria from hibernating and active ground squirrels, Citellus undulatus.

1. Electron microscopy of liver tissue preparations, obtained from hibernating ground squirrels, reveals mitochondria in a condensed state. 2. When kept on ice, mitochondria isolated from the livers of hibernating and active ground squirrels are in a shrunken state. 3. Incubation of mitochondria isolated from the livers of active ground squirrels in the presence of succinate, at 27 degrees C, results in mitochondrial swelling, while mitochondria from hibernating ground squirrels under the same conditions remain relatively shrunken. 4. The swollen mitochondria from active ground squirrels show high oxidative activity, while the shrunken mitochondria from hibernating animals show low oxidative activity. 5. Swelling of mitochondria from hibernating ground squirrels in a hypo-osmolar medium is accompanied by a significant increase in oxidative activity. 6. It is inferred that the shrinkage of hibernating ground squirrel mitochondria is one of the main causes of the inhibition of oxidative activity and other mitochondrial functions during hibernation.

Cyclosporin A suppression of uncoupling in liver mitochondria of ground squirrel during arousal from hibernation.

Energy coupling parameters were studied in liver mitochondria of ground squirrel during arousal from hibernation. It was found that such mitochondria become uncoupled during incubation with phosphate in a salt medium. The uncoupling was revealed by respiration rate increase and membrane potential decrease in the presence of oligomycin. Both effects were reversed by addition of cyclosporin A. Under the same in vitro conditions, mitochondria from aroused (active) animals showed no uncoupling but could be uncoupled by addition of palmitate in the cyclosporin A-sensitive fashion. It is proposed that formation of cyclosporin A-sensitive pores can be involved in urgent heat production in arousing hibernators.

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.

Regulation of oxidative activity and delta psi of liver mitochondria of active and hibernating gophers. The role of phospholipase A2.

1. In the present work the initially lowered oxidase activity of liver mitochondria of hibernating gophers is shown to increase upon Ca(2+)-loading, after freezing-thawing repeated three times and after swelling in a medium containing potassium acetate as well as in a hypotonic sucrose medium. 2. In all cases the inhibition of phospholipase A2 hindered the increase of the oxidase activity of mitochondria. 3. Mitochondria of hibernating gophers have a lowered delta psi in comparison with active animals, which is restored in the hypotonic medium.

Thermoregulatory, carboxyatractylate-sensitive uncoupling in heart and skeletal muscle mitochondria of the ground squirrel correlates with the level of free fatty acids.

Thermoregulatory uncoupling of oxidative phosphorylation has been studied in heart and skeletal muscle mitochondria of ground squirrels. The respiratory rate of mitochondria in the presence of oligomycin was found to be much higher in winter (in hibernating, arousing, or aroused animals) than in summer. This additional respiration is strongly (arousing animals) or completely (hibernating and aroused animals) inhibited by carboxyatractylate (CAtr) and bovine serum albumin (BSA). The CAtr- and BSA-induced decreases in the rate of respiration are accompanied by membrane potential increases. The rate of the CAtr- and BSA-sensitive respiration is proportional to the content of free fatty acids which, in the heart, decreases in the order: arousing greater than aroused = hibernating greater than summer animals. Maximal respiratory rates observed in the presence of dinitrophenol (arousing greater than aroused greater than summer greater than hibernating animals) do not parallel the fatty acid level. It is assumed that some heat production in the winter animals is due to fatty acid-induced, ATP/ADP-antiporter-mediated uncoupling in heart and skeletal muscle mitochondria. The peak of heat production during arousal after hibernation also includes some other stimulatory effect on mitochondrial respiration.

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.

Inhibitors of the ATP/ADP antiporter suppress stimulation of mitochondrial respiration and H+ permeability by palmitate and anionic detergents.

The action of ATP/ADP-antiporter inhibitors upon the uncoupling effect of palmitate, detergents and 'classical' uncouplers has been studied. The uncoupling effect was estimated by stimulation of succinate oxidation and of H+ permeability of rat liver mitochondria in the presence of oligomycin. It is shown that carboxyatractylate (CAtr) and pyridoxal 5-phosphate (PLP) suppress the uncoupling induced by palmitate and the anionic detergents SDS and cholate, but do not affect that induced by the cationic detergents CTAB, by the non-ionic detergent Triton X-100, as well as by the 'classical' uncouplers FCCP and DNP. The results are discussed in terms of a concept assuming that the ATP/ADP-antiporter facilitates the electrophoretic export of hydrophobic anions from mitochondria.

The role of phospholipase A2 in lipid peroxidation-induced fall of membrane potential of rat liver mitochondria.

Cumene hydroperoxide (230 microM)-induced fall of the membrane potential takes place only in Ca2(+)-loaded mitochondria. Inhibitor of phospholipase A2 p-bromphenacyl bromide prevents uncoupling of mitochondria, having no effect on the accumulation of lipid peroxidation products.

Reversible inhibition of electron transfer in the ubiquinol. Cytochrome c reductase segment of the mitochondrial respiratory chain in hibernating ground squirrels.

Electron transfer through the ubiquinol:cytochrome c1-segment of liver mitochondria isolated from hibernating ground squirrels Citellus undulatus is repressed by 70-80% as compared to mitochondria from the active animals. The inhibition site is likely to be localized between ubiquinone and the cytochrome bc1 complex. Partial release of the inhibition can be observed upon swelling of the isolated mitochondria in a hypoosmotic medium, the effect being prevented by phospholipase A2 inhibitors. Possible role of phospholipase A2 in regulation of ubiquinol oxidation by complex bc1 is discussed.

Regulation of the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels, Citellus undulatus.

1. The rates of oxidation of various substrates (beta-hydroxybutyrate, succinate, ascorbate + TMPD) and the rate of ATP synthesis in liver mitochondria from active and hibernating ground squirrels were measured. 2. It was shown that the rate of mitochondrial respiration is significantly lower in hibernating animals than in active animals. 3. The degree of inhibition of mitochondrial respiration in hibernating ground squirrels was found to correlate with the length of the respiratory chain fragment involved in the oxidation of a given substrate. 4. The inhibition of mitochondrial respiration in hibernating animals was accompanied by a decrease in the rate of ATP synthesis. 5. The activity of phospholipase A2 in liver mitochondria from hibernating ground squirrels was found to be decreased. The activation of phospholipase A2 by Ca2+ ions eliminated the inhibition of respiration almost completely. 6. It was assumed that the inhibition of mitochondrial respiration during hibernation is (a) related to the suppression of phospholipase A2 activity and (b) caused by the reduced rates of electron transport through the respiratory chain and/or of substrate transport across the mitochondrial membrane.

Role of the Ca2+ cycle in uncoupling of oxidative phosphorylation in liver mitochondria of cold-acclimated rats.

Cold acclimation of Wistar rats for 2-4 weeks at about 3 degrees C resulted in an increased respiration rate and a reduced ADP/O ratio in liver mitochondria. With increasing duration of acclimation up to 10-12 weeks, these parameters returned to a normal level. The increase in the respiration rate and the decline of the mitochondrial ADP/O ratio were associated with a significant activation of the electroneutral release of Ca2+. When the animals were acclimated for 10-12 weeks the rate of Ca2+ release reduced to control values. The addition of 1 microM ruthenium red resulted in a decrease in the rates of mitochondrial respiration in control and cold-acclimated rats to approximately equal values and in a partial restoration of the ADP/O ratio in liver mitochondria of rats kept in the cold for 2-4 weeks. The respiratory activity of mitochondria isolated in the presence of 1 mM EGTA unaffected by ruthenium red.