antimycin and carboxyatractyloside

The aim of our study was to analyze a distribution of metabolic flux controls of all mitochondrial complexes of ATP-Synthasome and mitochondrial creatine kinase (MtCK) in situ in permeabilized cardiac cells. For this we used their specific inhibitors to measure flux control coefficients (C(vi)(JATP)) in two different systems: A) direct stimulation of respiration by ADP and B) activation of respiration by coupled MtCK reaction in the presence of MgATP and creatine. In isolated mitochondria the C(vi)(JATP) were for system A: Complex I - 0.19, Complex III - 0.06, Complex IV 0.18, adenine nucleotide translocase (ANT) - 0.11, ATP synthase - 0.01, Pi carrier - 0.20, and the sum of C(vi)(JATP) was 0.75. In the presence of 10mM creatine (system B) the C(vi)(JATP) were 0.38 for ANT and 0.80 for MtCK. In the permeabilized cardiomyocytes inhibitors had to be added in much higher final concentration, and the following values of C(vi)(JATP) were determined for condition A and B, respectively: Complex I - 0.20 and 0.64, Complex III - 0.41 and 0.40, Complex IV - 0.40 and 0.49, ANT - 0.20 and 0.92, ATP synthase - 0.065 and 0.38, Pi carrier - 0.06 and 0.06, MtCK 0.95. The sum of C(vi)(JATP) was 1.33 and 3.84, respectively. Thus, C(vi)(JATP) were specifically increased under conditions B only for steps involved in ADP turnover and for Complex I in permeabilized cardiomyocytes within Mitochondrial Interactosome, a supercomplex consisting of MtCK, ATP-Synthasome, voltage dependent anion channel associated with tubulin βII which restricts permeability of the mitochondrial outer membrane.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Atractyloside; Cell Respiration; Creatine Kinase, Mitochondrial Form; Dinitrofluorobenzene; Energy Metabolism; Enzyme Inhibitors; Male; Mersalyl; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Proton-Translocating ATPases; Models, Theoretical; Myocytes, Cardiac; Oxygen Consumption; Rats; Rats, Wistar; Rotenone; Sodium Cyanide; Uncoupling Agents

The respiratory capacities of hepatocytes, derived from hypothyroid, euthyroid and hyperthyroid rats, have been compared by measuring rates of oxygen uptake and by titrating components of the respiratory chain with specific inhibitors. Thyroid hormone increased the maximal rate of substrate-stimulated respiration and also increased the degree of ionophore-stimulated oxygen uptake. In titration experiments, similar concentrations of oligomycin or antimycin were required for maximal inhibition of respiration regardless of thyroid state, suggesting that the changes in respiratory capacity were not the result of variation in the amounts of ATP synthase or cytochrome b. However, less rotenone was required for maximal inhibition of respiration in the hypothyroid state than in cells from euthyroid or hyperthyroid rats, implying that hepatocytes from hypothyroid animals contain less NADH dehydrogenase. The concentration of carboxyatractyloside necessary for maximal inhibition of respiration was 100 microM in hepatocytes from hypothyroid rats, but 200 microM and 300 microM in hepatocytes from euthyroid and hyperthyroid rats, respectively, indicating a possible correlation between levels of thyroid hormone and the amount or activity of adenine nucleotide translocase. The increased capacity for coupled respiration in response to thyroid hormone is not associated with an increase in the components of the electron transport chain or ATP synthase, but correlates with an increased activity of adenine nucleotide translocase.

Topics: Animals; Antimycin A; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Hyperthyroidism; Hypothyroidism; Kinetics; Liver; Male; Oligomycins; Oxygen Consumption; Rats; Rats, Inbred Strains; Reference Values; Rotenone; Thyroid Gland; Triiodothyronine

The ability of isolated rat heart mitochondria to take up coenzyme A (CoA) from the incubation medium was studied. Mitochondria accumulated CoA in a time- and concentration-dependent manner. The accumulation process occurred in two phases. Within the first 30 s of incubation, mitochondrial content of CoA increased, and this phase did not plateau in the concentration range studied. Following this initial increase, a second slower phase of CoA accumulation occurred which plateaued around 50 microM CoA. The initial phase was decreased significantly by ATP or by carboxyatractyloside. In contrast, the presence of ATP or carboxyatractyloside did not affect the second phase. Decreasing the temperature from 30 to 0 degrees C did not affect the initial phase, but the second phase was almost abolished. In the presence of metabolic inhibitors (either 2,4-dinitrophenol or a combination of rotenone and antimycin), the initial "binding" phase was not affected; but the second "uptake" phase was abolished. These results suggest that the first phase of mitochondrial CoA accumulation is probably CoA binding to adenine recognizing sites on the mitochondria while the second phase may represent a specific uptake process for CoA which, although not directly ATP-dependent, is energy-dependent.

Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Animals; Antimycin A; Atractyloside; Biological Transport, Active; Coenzyme A; Dinitrophenols; Male; Mitochondria, Heart; Rats; Rats, Inbred Strains; Rotenone; Temperature

Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; In Vitro Techniques; Mitochondria; NAD; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Protons; Rats; Succinates; Succinic Acid; Thermodynamics