Features of the uncoupling effect of fatty acids in liver mitochondria of mammals with different body weight.

In the presence of oligomycin, EGTA, and magnesium ions, the protonophore uncoupling activity of palmitate (V(Pal)) is determined as the ratio of the acceleration of respiration with palmitate to its concentration. Under these conditions, V(Pal) in liver mitochondria of one-month-old rats with the body weight of 50 g is 1.46-fold higher than in liver mitochondria of adult rats with the body weight of 250 g, whereas the uncoupling activity of FCCP does not depend on the age of the animals. The difference in V(Pal) is mainly due to its component insensitive to carboxyatractylate and glutamate (V(Ins)). This value is 2.9-fold higher in mitochondria of one-month-old rats than in those of adult rats. The protonophore activity of palmitate is similar in liver mitochondria of four-day-old and adult rats. In liver mitochondria of adult mammals (mouse, rat, guinea pig, rabbit), V(Pal) decreases with increase in the body weight of the animals. In double logarithmic coordinates, the dependence of the V(Pal) value on the body weight is linear with slope angle tangent of -0.18. The V(Pal) value is mainly contributed by its component V(Ins). In the presence of calcium ions, palmitate induces the nonspecific permeability of the inner membrane of liver mitochondria (pore opening). This Ca2+-dependent uncoupling effect of palmitate is less pronounced in mitochondria of one-month-old rats than in those of adult rats. In mitochondria of adult animals (mice, rats, and guinea pigs), the Ca2+-dependent uncoupling activity of palmitate is virtually the same. It is concluded that the protonophore uncoupling effect of palmitate in liver mitochondria of mammals, unlike its Ca2+-dependent effect, is associated with thermogenesis at rest and also with production of additional heat on cooling of the animals.

Temperature dependence of rat liver mitochondrial respiration with uncoupling of oxidative phosphorylation by fatty acids. Influence of inorganic phosphate.

The respiration rate of liver mitochondria in the course of succinate oxidation depends on temperature in the presence of palmitate more strongly than in its absence (in state 4). In the Arrhenius plot, the temperature dependence of the palmitate-induced stimulation of respiration has a bend at 22 degrees C which is characterized by transition of the activation energy from 120 to 60 kJ/mol. However, a similar dependence of respiration in state 4 is linear over the whole temperature range and corresponds to the activation energy of 17 kJ/mol. Phosphate partially inhibits the uncoupling effect of palmitate. This effect of phosphate is increased on decrease in temperature. In the presence of phosphate the temperature dependence in the Arrhenius plot also has a bend at 22 degrees C, and the activation energy increases from 128 to 208 kJ/mol in the range from 13 to 22 degrees C and from 56 to 67 kJ/mol in the range from 22 to 37 degrees C. Mersalyl (10 nmol/mg protein), an inhibitor of the phosphate carrier, similarly to phosphate, suppresses the uncoupling effect of laurate, and the effects of mersalyl and phosphate are not additive. The recoupling effects of phosphate and mersalyl seem to show involvement of the phosphate carrier in the uncoupling effect of fatty acids in liver mitochondria. Possible mechanisms of involvement of the phosphate carrier in the uncoupling effect of fatty acids are discussed.

Effect of ethanol on the palmitate-induced uncoupling of oxidative phosphorylation in liver mitochondria.

The effect of ethanol on the uncoupling activity of palmitate and recoupling activities of carboxyatractylate and glutamate was studied in liver mitochondria at various Mg2+ concentrations and medium pH values (7.0, 7.4, and 7.8). Ethanol taken at concentration of 0.25 M had no effect on the uncoupling activity of palmitic acid in the presence of 2 mM MgCl2 and decreased the recoupling effects of carboxyatractylate and glutamate added to mitochondria either just before or after the fatty acid. However, ethanol did not modify the overall recoupling effect of carboxyatractylate and glutamate taken in combination. The effect of ethanol decreased as medium pH was decreased to 7.0. Elevated concentration of Mg2+ (up to 8 mM) inhibits the uncoupling effect of palmitate. Ethanol eliminates substantially the recoupling effect of Mg2+ under these conditions, but does not influence the recoupling effects of carboxyatractylate and glutamate. It is inferred that ADP/ATP and aspartate/glutamate antiporters are involved in uncoupling function as single uncoupling complex with the common fatty acid pool. Fatty acid molecules gain the ability to migrate under the action of ethanol: from ADP/ATP antiporter to aspartate/glutamate antiporter on addition of carboxyatractylate and in opposite direction on addition of glutamate. Possible mechanisms of fatty acid translocation from one transporter to another are discussed.

Role of the ADP/ATP and aspartate/glutamate antiporters in the uncoupling effect of fatty acids, lauryl sulfate, and 2, 4-dinitrophenol in liver mitochondria.

Study of the uncoupling effect of various saturated fatty acids (from caprylic to palmitic) revealed that the glutamate recoupling effect was more pronounced in the case of short chain fatty acids, whereas recoupling of mitochondria by carboxyatractylate was more effective in the case of long chain fatty acids. The overall recoupling effect, however, did not depend on the fatty acid chain length. Besides carboxyatractylate, glutamate and aspartate also exhibited a recoupling effect under uncoupling by lauryl sulfate. The uncoupling effect of lauryl sulfate was markedly weaker in the presence of DNP or laurate (but not FCCP) which were added in concentrations causing twofold increase in mitochondrial respiration. In the presence of lauryl sulfate the uncoupling action of laurate and DNP was insensitive to carboxyatractylate and glutamate. With laurate and DNP as uncouplers increasing the pH from 7.0 to 7.8 potentiated the recoupling effect of carboxyatractylate and attenuated the recoupling effect of glutamate. In the case of uncoupling by lauryl sulfate similar changes in the recoupling effect of carboxyatractylate and glutamate were observed only in the presence of 10 microM tetraphenylphosphonium. Thus, when uncoupling is induced by fatty acids, DNP, and lauryl sulfate, the ADP/ATP and aspartate/glutamate antiporters function as two parallel and independent pathways for mitochondrial membrane potential dissipation. We suggest that the role of the ADP/ATP antiporter in uncoupling includes proton capture from the intermembrane space with subsequent protonation of uncoupler anions, their transport as neutral molecules on the internal side, and deprotonation followed by proton release into the matrix and transfer of the uncoupler anion in the reverse direction. During uncoupling the aspartate/glutamate antiporter cyclically carries the uncoupler anion with simultaneous proton transfer from the intermembrane space into the matrix.

Involvement of aspartate/glutamate antiporter in uncoupling effect of fatty acids in heart mitochondria.

Earlier it was shown that fatty acid-induced uncoupling in liver mitochondria is suppressed by the substrates of the aspartate/glutamate antiporter (V. N. Samartsev, A. V. Smirnov, I. P. Zeldi, O. V. Markova, E. N. Mokhova, and V. P. Skulachev (1997) Biochim. Biophys. Acta, 1319, 251-257). In this study it is shown that in heart mitochondria aspartate, glutamate, and diethyl pyrocarbonate do not affect oxygen consumption and membrane potential in the presence of laurate at pH 7.4. These compounds have a weak (versus carboxyatractylate) coupling effect at pH 7.0. This effect is manifested only in the presence of carboxyatractylate, magnesium, and phosphate in the incubation medium. It is suggested that these tissue-specific effects are due not only to the specific characteristics of aspartate/glutamate antiporter, but also to the differences in the content of endogenous metabolites in heart mitochondria.

Involvement of aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria.

Effects of aspartate, glutamate and an inhibitor of the aspartate/glutamate antiporter, diethylpyrocarbonate (DEPC), on uncoupling of the energy transduction processes in rat liver mitochondria have been investigated. It is found that both the antiporter substrates and the antiporter inhibitor operate as recouplers when uncoupling is caused by free fatty acids (FFA). Recoupling consists in (1) partial inhibition of the FFA-stimulated respiration and (2) some increase in the membrane potential. Half-maximal effects are observed at concentrations of glutamate and aspartate close the K(m) values of the antiporter. Recouplings by glutamate (aspartate) and DEPC are not additive. On the other hand, recoupling by any of these compounds and carboxyatractylate or ADP appears to be additive. Uncoupling by dinitrophenol is less sensitive to the recouplers whereas that by FCCP is not sensitive at all. It is concluded that uncoupling by FFA in rat liver mitochondria is mediated not only by the ATP/ADP antiporter but also by the aspartate/glutamate antiporter.

[The effect of p-chloromercuribenzoate on regulation of oxidative phosphorylation by ADP and ATP and stimulation of liver mitochondrial respiration by palmitate]. / Vliianie p-khloromerkuribenzoata na reguliatsiiu ADP i ATP okislitel'nogo fosforilirovaniia i stimuliruemogo pal'mitatom dykhaniia mitokhondrii pecheni.

The effect of p-chloromercuribenzoate (pCMB) on oxidative phosphorylation and palmitate-stimulated respiration of liver mitochondria has been studied. pCMB (1 microM) does not affect oxidative phosphorylation but reduces the inhibiting effect of ATP on this process. Used at the same concentration, pCMB eliminates the inhibiting effect of ADP and ATP on mitochondrial respiration in the presence of 10 and 20 microM palmitate. pCMB has no effect on inhibition of oxidative phosphorylation by carboxyatractyloside or on palmitate-stimulated respiration of mitochondria. It is concluded that the SH-groups of mitochondria localized in the hydrophilic region outside the inner mitochondrial membrane participate in regulation of oxidative phosphorylation by ATP and in regulation of palmitate-stimulated respiration by ADP + ATP.

[Participation of SH-groups in regulating oxidative phosphorylation by malate and palmitate-uncoupled respiration in liver mitochondria]. / Uchastie SH-grupp v reguliatsii malatom okislitel'nogo fosforilirovaniia i razobshchennogo pal'mitatom dykhaniia v mitokhondriiakh pecheni.

The effect of malate on respiration in liver mitochondria has been studied during oxidation of succinate in the presence of rotenone both in state 3 and after palmitate addition. Malate was shown to stimulate the rate of mitochondrial respiration in the both respiratory states, its effect being increased in the presence of the NAD-dependent substrates of oxidation-glutamate and pyruvate or thiols (cysteine and thiourea) Preincubation of mitochondria for 5 min in the absence of respiratory substrates eliminated the stimulating effect of malate. However, this effect was manifested in the conditions when the NAD-dependent respiratory substrates or thiols were added after preincubation of mitochondria. p-Chloromercuribenzoate eliminated the stimulating effect of malate. Carboxyatractyloside and ATP inhibited mitochondrial respiration in the presence of palmitate. Malate did not influence the action of the first effector but eliminated that of the second effector. It is concluded that malate can regulate oxidative phosphorylation and palmitate-uncoupled respiration by affecting the adenine nucleotide transported. The SH-groups localized outside the mitochondria in the hydrophilic region play an important role in the realization of malate effects.

[Age peculiarities in the content of nucleic acids, collagen (as oxyproline) and other proteins in rat skin and lung tissues]. / Vozrastnye osobennosti v soderzhanii nukleinovykh kislot, kollagena (po oksiprolinu) i drutikh belkov v tkaniakh kozhi i lerkikh krys.

It is established that in old animals, as compared to the young ones, the content of nucleic acids, the total content of protein and the content of the peptide-bound oxyproline in the collagen soluble fractions in the skin and lungs tissues decrease, whereas the content of peptide-bound oxyproline in the insoluble collagen and of free oxyproline, vice versa, increases.