Isolated Rabbit Heart Mitochondria Research Articles

Effect of the mitochondrial membrane potential on the absorbance of the reduced form of cytochrome c oxidase

The effect of mitochondrial membrane potential (ΔΨm) on the absorbance of the reduced cytochrome c oxidase (COX) was evaluated in isolated rabbit heart mitochondria using integrating sphere optical spectroscopy. Maximal reduction of the mitochondrial cytochromes was achieved by either blowing nitrogen to remove oxygen, or by adding cyanide. Gradual depolarization of ΔΨm by adding increasing concentrations of uncoupler resulted in an increase of up to 50 % in the absorbance of cytochrome aa3 under nitrogen saturation, and of 25 % with cyanide. Cytochrome aa3 absorbance increases were also observed in the presence of cyanide with apyrase (20 %) or oligomycin (12 %). The bL heme absorbance also decreased as expected from ΔΨm depolarization. A ~ 1 nm red shift in the peak wavelength of cytochrome aa3 was observed under anoxic conditions as ΔΨm was depolarized. Importantly, cytochrome c and c1 absorbances remained constant at levels corresponding to full reduction under all experimental manipulations of ΔΨm, especially with cyanide. These data suggest that ΔΨm-dependent changes in the absorbance of reduced COX were due to a variable extinction coefficient of heme a and/or a3 as a function of ΔΨm. A similar increase in the reduced cytochrome aa3 absorbance without changes in cytochrome c and c1 was observed in the perfused rabbit heart when decreasing ΔΨm with uncoupler. Our results imply that COX absorbance in its fully reduced state does not simply reflect the oxygen tension but also the ΔΨm. This may prove useful in monitoring ΔΨm under anoxic or ischemic conditions in intact tissue.

Fatty acid composition of mitochondrial phospholipids of the ischemic heart

Gas liquid chromatography was used to study fatty acid (FA) composition of lysophosphatidylcholine (lyso-PC) and phosphatidylcholine in isolated rabbit heart mitochondria. In control, lyso-PC and PC were found to contain 95 and 66% of unsaturated FA, respectively. At 1 hour of ischemia (autolysis at 37 degrees C) the percentage of saturated FA in lyso-PC noticeably increased whereas FA composition remained unchanged. It is concluded that changes in FA composition of lyso-PC are caused by phospholipase A2 action.

Selective inhibition of Na+-induced Ca2+ release from heart mitochondria by diltiazem and certain other Ca2+ antagonist drugs.

The effect of Ca2+ antagonist drugs on Ca2+ uptake and Na+-induced Ca2+ release by isolated rabbit heart mitochondria has been studied using arsenazo III and dansylaziridine-labeled troponin C as Ca2+ indicators. Mitochondria accumulated 20 nmol of Ca2+/mg of protein by a respiration-dependent process. A23187, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or Na+ induced a release of Ca2+ from Ca2+-loaded and ruthenium red (RR)-treated mitochondria. Ca2+ antagonist drugs had no effect on the Ca2+ uptake process or on Ca2+ release induced either by A23187 or by carbonyl cyanide p-trifluoromethoxyphenylhydrazone but they were effective in inhibiting the Na+-induced Ca2+ release process. The drug concentrations that resulted in 50% inhibition of Na+-induced Ca2+ release were 7, 12, 13, 66, and 150 microM for diltiazem, prenylamine, fendiline, nifedipine, and verapamil, respectively. In the absence of RR, the net amount of Ca2+ released by Na+ was less than that which occurred in the presence of RR due to the simultaneous operation of both the Ca2+ uptake and the Ca2+ release processes. Under these conditions, the inhibition of Ca2+ uptake by RR brought about a complete release of Ca2+ from the mitochondria, and the inhibition of the Na+-induced Ca2+ release by diltiazem resulted in an apparent uptake of Ca2+ by the mitochondria. These results indicate that diltiazem and certain other Ca2+ antagonist drugs may be selective inhibitors of the Na+/Ca2+ antiporter in heart mitochondria much like ruthenium red is a selective inhibitor of the Ca2+ uniporter.

Perinatal changes in mitochondrial respiration of the rabbit heart.

Oxidative metabolism and citrate synthase activity during the perinatal period were assessed in isolated rabbit heart mitochondria at 27 days' gestation (27 dpc), 1 day postpartum (1 dpp) and 10 dpp. Oxygen consumption was measured during state 3 and state 4 respiration using the following substrates: glutamate, alpha-ketoglutarate, pyruvate/malate, succinate/rotenone, acetate, palmitylcarnitine, and palmityl CoA/carnitine. Results obtained from fetal and neonatal animals were compared to values obtained from adult left and right ventricles. Rates of state 3 oxygen consumption per milligram mitochondrial protein using tricarboxylic acid cycle intermediates or palmitylcarnitine were higher in both 1 and 10 dpp animals than in either left or right ventricles. Respiratory activity per milligram mitochondrial protein using these substrates was similar in 27 dpc fetuses and adult left ventricles. The rate of oxidation of palmityl CoA was lower in fetal mitochondria than in adult left ventricle, indicating that the capacity for conversion of long-chain fatty acyl CoA to acylcarnitine necessary for transport of fatty substrates into the mitochondria is decreased in fetal hearts. Increases in myocardial citrate synthase corresponded to increases in mitochondrial mass reported by other investigators.

Intramitochondrial adenine nucleotides and energy-linked functions of heart mitochondria.

Isolated rabbit heart mitochondria were incubated with varying amounts of inorganic pyrophosphate in 250 mM sucrose to specifically decrease the pool size of endogenous adenine nucleotides. The endogenous adenine nucleotide content decreased by as much as 80% as a result of this treatment. Phosphorylating respiration (state 3) declined from about 340 to 180 nAtoms O . min-1 . mg protein-1 over the full range of intramitochondrial adenine nucleotides measured (approx 7.5-1.5 nmol/mg protein). Uncoupled and nonphosphorylating (state 4) rates of respiration were not greatly affected by adenine nucleotide depletion. Respiratory activity of the adenine nucleotide-depleted mitochondria was enhanced by addition of exogenous adenosine 5'-triphosphate (ATP). Partial depletion (approx 40%) of the intramitochondrial adenine nucleotides resulted in an impaired ability of heart mitochondria to retain Ca2+. Premature Ca2+ efflux was associated with organelle swelling and altered energy coupling. Exogenous ATP or adenosine 5'-diphosphate (ADP) added prior to Ca2+ efflux restored Ca2+ retention in these mitochondria. Atractyloside inhibited the restoration of Ca2+ retention. This study indicates a significant role for endogenous adenine nucleotides in maintaining oxidative phosphorylation and Ca2+ transport in heart mitochondria. The results are discussed with regard to significance in ischemic heart damage.

The lack of direct coupling between ATP-ADP translocase and creatine phosphokinase in isolated rabbit heart mitochondria

The formation of creatine phosphate by isolated rabbit heart mitochondria in the presence of creatine, α-ketoglutarate, ATP, and inorganic phosphate was studied. Creatine phosphate formation was inhibited by oligomycin. This was most probably due to increased concentration of ADP favoring the reverse reaction (formation of creatine and ATP from phosphocreatine and ADP). The inhibitory effect of oligomycin disappeared in the presence of phosphoenolpyruvate and pyruvate kinase. The results do not indicate any direct coupling between mitochondrial creatine phosphokinase and ATP-ADP translocase as has been suggested for rat heart mitochondria.

Protection of oxidative phosphorylation by bencyclane against the damaging effect of mitochondrial swelling

The effect of bencyclane on the relationship between the energy-dependent swelling and oxidative phosphorylation was studied in isolated rabbit heart mitochondria. The oxygen consumption and the osmotic volume changes of mitochondria as well as the extramitochondrial proton concentration were simultaneously measured in the same sample. In sucrose medium the oxidative phosphorylation was uncoupled and the electron transport chain inhibited by bencyclane concentrations higher than 10 μM. In a medium of high potassium concentration the swelling of mitochondria was accompanied both by a decrease rate and efficiency of oxidative phosphorylation. Both swelling and impairment of oxidative phosphorylation were inhibited by 2.5–10 μM bencyclane concentrations. The relation of these effects to the myocardial function was discussed.

The regulation of pyruvate dehydrogenase by fatty acids in isolated rabbit heart mitochondria

The rate of pyruvate oxidation by isolated rabbit heart mitochondria was inhibited by fatty acylcarnitine derivatives. The extent of inhibition by pyruvate oxidation in State 3 was greatest with palmitylcarnitine and only a minimal inhibition was observed with acetylcarnitine, while octanoylcarnitine or octanoate caused an intermediate extent of inhibition. Analyses of the intramitochondrial ATP ADP and NADH NAD + ratios under the different conditions of incubation indicated that it is unlikely that changes in either or both of these parameters were the primary negative effectors of the rate of pyruvate oxidation. A positive correlation between the decrease in the rate of pyruvate oxidation and the decrease in the level of free CoASH in the mitochondria was observed. Extraction and assay of the pyruvate dehydrogenase from rabbit heart mitochondria during the time course of the fatty acid-mediated inhibition of pyruvate oxidation indicated that pyruvate dehydrogenase was strongly inactivated when palmitylcarnitine was the fatty acid, while incubation with octanoate and acetylcarnitine resulted in less extensive inactivation of pyruvate dehydrogenase. Measurement of the effects of NADH, NAD +, acetyl-CoA, and CoASH on the inactivation of pyruvate dehydrogenase extracted from rabbit heart mitochondria indicated that NADH and acetyl-CoA activated the pyruvate dehydrogenasee kinase while CoASH strongly inhibited the kinase and NAD + was without effect. In addition, palmityl-CoA and octanoyl-CoA had little, if any, effect on the pyruvate dehydrogenase kinase activity. It was observed that palmityl-CoA but not octanoyl-CoA strongly inhibited the activity of the extracted pyruvate dehydrogenase. Hence, it is concluded that (a) decreased mitochondrial CoASH levels, which essentially remove a potent inhibitor of the pyruvate dehydrogenase kinase, (b) possibly a diminished free CoASH supply, which may be utilized as a substrate for the active complex, and (c) direct inhibitory effects of palmityl-CoA on the active form of the pyruvate dehydrogenase complex combine to make palmitylcarnitine a much more potent inhibitor of mitochondrial pyruvate oxidation than shorter chain length acylcarnitine derivatives.

Formation of creatine phosphate from creatine and 32P-labelled ATP by isolated rabbit heart mitochondria

With ATP [γ- 32P] we have demonstrated directly that mitochondrial creatine phosphokinase catalyzes the formation of large amounts of creatine phosphate with mitochondria generated ATP as substrate rather than added extramitochondrial ATP.

Fatty acid synthesis and Krebs' cycle activity in heart mitochondria

The relationship between [1- 14C]acetate incorporation into fatty acids (FA) and the concomitant [1- 14C]acetate flux through citrate synthase (CS) was studied in isolated rabbit heart mitochondria during state 4 (Chance) respiration. The flux through CS was calculated from the 14C content of 14CO 2, Krebs' cycle acids, and glutamate; the latter were extracted and separated by ion exchange chromatography. Both succinate in low concentration and l-malate effected low NADH NAD ratios and increased flux through CS without stimulating incorporation into FA. Succinate in high concentration effected high NADH NAD ratios, reduced flux through CS, and stimulated incorporation into FA. In the presence of succinate both rotenone and l-carnitine inhibited incorporation into FA but not flux through CS despite high NADH NAD ratios. Maximal [2- 14C]pyruvate flux through CS approximated that of [ 14C]acetate. However, with the high NADH NAD ratio effected by succinate, the [2- 14C]pyruvate flux through CS was greater than that of [ 14C]acetate and the [2- 14C]pyruvate incorporation into FA was only about one-tenth that of [ 14C]acetate. Studies with the isolated perfused rabbit heart showed that hypoxia increased five-to-nine fold the net [U- 14C]glucose incorporation into FA. We conclude that in heart mitochondria a high NADH NAD ratio not only stimulates [ 14C]acetate incorporation into FA but also concomitantly slows flux through citrate synthase by decreasing the oxalacetate level. The data suggest an acetyl CoA pool available for FA synthesis different from that for CS. During myocardial hypoxia mitochondrial FA synthesis may supplement lactate production as a mechanism for NADH oxidation.

Regulation of Pyruvate Oxidation in Isolated Rabbit Heart Mitochondria

Abstract Evidence is presented in support of the hypothesis that the pyruvate dehydrogenase multienzyme complex of intact rabbit heart mitochondria may be regulated by a phosphorylation-dephosphorylation mechanism. Mitochondria incubated in the presence of pyruvate plus l-malate and either ADP or uncoupler exhibit nearly identical, rapid rates of pyruvate oxidation but possess markedly different ATP levels. It was shown that under metabolic conditions which lead to a release or mobilization of intramitochondrial magnesium and which also have a high intramitochondrial ATP level, pyruvate oxidation was nearly completely inhibited after a brief lag phase. It was shown that the addition of exogenous magnesium to uncoupled mitochondria supplemented with ADP caused a more rapid inhibition of pyruvate oxidation. The observed inhibition of pyruvate oxidation was dependent upon the time of preliminary incubation with uncoupler and ADP and was atractyloside-sensitive. Evidence was obtained indicating that the inhibition of pyruvate oxidation was specific for the substrate, pyruvate, i.e. the oxidation of l(-)palmitylcarnitine or α-ketoglutarate was unaffected under conditions leading to an inhibition of pyruvate oxidation. In addition, it was demonstrated that the inhibition of pyruvate oxidation was not caused by an accumulation of either NADH or acetyl-CoA, both inhibitory products of the pyruvate dehydrogenase reaction, during the course of these experiments. The experiments reported in this communication indicate that the control of the availability of both ATP and magnesium are crucial for the regulation of the pyruvate dehydrogenase multienzyme complex. These studies are consistent with the possibility that the pyruvate dehydrogenase-linked protein kinase may be an effective means of regulating the conversion of pyruvate to acetyl-CoA in intact metabolic systems such as the isolated mitochondrion.