oligomycins and Coronary-Disease

The effect of inhibition of the mitochondrial ATPase with oligomycin on the rate of ATP depletion and anaerobic glycolysis was studied in the totally ischemic dog heart. An oxygenated, buffered crystalloidal solution containing 10 microM oligomycin and 12 mM glucose was delivered at 100 mmHg pressure to the circumflex bed of the excised cooled heart. Buffered solution without oligomycin was delivered simultaneously to the anterior descending bed of the same heart. Little metabolic evidence of ischemia developed until the heart was made totally ischemic by incubating it in a sealed plastic bag at 37 degrees C. Successful inhibition of the mitochondrial ATPase was confirmed by the absence of both mitochondrial ATPase activity and the loss of respiratory control in mitochondria isolated from treated tissue. ATP, glycolytic intermediates and catabolites of the adenine nucleotide pool were measured in the control and treated beds at various intervals during 120 min of ischemia. Inhibition of the ATPase resulted in slowing of the rates of ATP depletion and anaerobic glycolysis (estimated by lactate accumulation). Also, degradation of the adenine nucleotide pool occurred more slowly in the inhibited group. These data establish that about 35% of the ATP utilization observed during the first 90 min of total ischemia in the canine heart is due to mitochondrial ATPase activity.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Anaerobiosis; Animals; Coronary Disease; Dogs; Glycolysis; In Vitro Techniques; Mitochondria, Heart; Oligomycins; Perfusion

In the present study, isolated dog and rat hearts were perfused in the Langendorff mode with Krebs bicarbonate buffer in the absence and presence of 10(-5) M oligomycin. The perfusion protocols employed allowed tissue pH to drop during subsequent ischemic incubations essentially as it would in blood-perfused hearts. Tissue pH, ATP, lactate, and mitochondrial respiratory function were measured during the course of subsequent zero-flow ischemic incubations. The adenosinetriphosphatase (ATPase) activities attributable to both mitochondrial and nonmitochondrial ATPases in sonicated heart homogenates and the actomyosin ATPase in isolated cardiac myofibrils were measured in both species. Consistent with earlier results with a different model in which tissue pH was buffered during the ischemic incubations [W. Rouslin, J. L. Erickson, and R. J. Solaro. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): H503-H508, 1986], the inhibition of the mitochondrial ATPase in situ by oligomycin markedly slowed both tissue ATP depletion and the loss of mitochondrial function during ischemia in the dog. However, oligomycin had only a very small and transient effect on ATP depletion and mitochondrial function in the rat. This was apparently so because of the fivefold higher rate of glycolytic ATP production as well as the nearly threefold higher total nonmitochondrial ATPase activity of ischemic rat compared with ischemic dog heart. These results suggest that although the inhibition of the mitochondrial ATPase makes a major contribution to ATP conservation in ischemic dog heart, it makes only a very small contribution in rat.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Coronary Disease; Dogs; Female; Heart Rate; Hydrogen-Ion Concentration; Lactates; Lactic Acid; Male; Mitochondria, Heart; Myocardium; Oligomycins; Oxygen Consumption; Rats

In the present study we examined factors affecting the reversal of the ischemia-induced protonic inhibition of the mitochondrial ATPase described earlier (Rouslin, W. (1983) J. Biol. Chem. 258, 9657-9661). It was found that ATPase reactivation and accompanying inhibitor protein release during the re-energization of intact mitochondria isolated from 20-min ischemic canine heart muscle could be blocked completely by either carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or nigericin but was unaffected by valinomycin at 35 mM K+. At higher K+ concentrations, valinomycin also blocked ATPase reactivation but not quite as completely as did nigericin. These observations suggest that ATPase reactivation and inhibitor protein release are particularly dependent upon either the trans-inner membrane pH gradient (delta pH) or possibly upon matrix pH per se and slightly less dependent upon membrane potential (delta psi) in intact cardiac muscle mitochondria. The addition of FCCP at the end of the re-energization incubations limited partially the extent of both ATPase reactivation and inhibitor protein release. This latter effect appears to have been mediated by a partial reassociation of the inhibitor protein with the enzyme, and it was accentuated (when FCCP was added at the end of the incubations) or mimicked (when FCCP was absent) by lowering the pH of the re-energization medium. A close examination of the first 10 min of the time course of enzyme activation and of inhibitor protein release revealed that while the former process was essentially finished in 1 min or less, the latter required approximately 10 min for completion. This observation led to the proposal of a two-site model of enzyme-inhibitor interaction which is discussed.

Topics: Adenosine Triphosphatases; Animals; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Coronary Disease; Dogs; Enzyme Activation; Female; Hydrogen-Ion Concentration; Kinetics; Male; Mitochondria, Heart; Nigericin; Oligomycins; Potassium; Proteins; Submitochondrial Particles; Valinomycin

The perfusion of canine cardiac muscle with 10 microM oligomycin produced a nearly 90% slowing of the net rate of tissue ATP depletion from 0.200 to 0.025 mumol X min-1 X g wet wt-1 of tissue during a subsequent myocardial autolytic interval during which tissue pH was held constant. Moreover, lowering the tissue pH during the autolytic process by 0.6 unit from approximately 6.8 to approximately 6.2 produced a nearly 60% slowing of the net rate of tissue ATP depletion from 0.200 to 0.087 mumol X min-1 X g wet wt-1. The pH dependence of the net rate of tissue ATP depletion (by an oligomycin-sensitive process) was that predicted from the mitochondrial ATPase pH-inhibition profiles reported earlier (J. Biol. Chem. 258: 9657-9661, 1983). When taken together with our observation that the mitochondrial ATPase comprises approximately 90% of the total of all of the ATP hydrolyzing activities present in cardiac muscle cells, data reported here suggest that the protonic inhibition of the mitochondrial ATPase plays a major role in regulating the rate of tissue ATP depletion during myocardial ischemia.

Topics: Acidosis; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Autolysis; Coronary Disease; Dogs; Female; HEPES; Hydrogen-Ion Concentration; Male; Mitochondria, Heart; Myocardium; Myofibrils; Oligomycins

The present study utilized a cultured myocardial cell model to evaluate the relationship between the release of arachidonate from membrane phospholipids, and the progression of cell injury during ATP depletion. High-energy phosphate depletion was induced by incubating cultured neonatal rat myocardial cells with various combinations of metabolic inhibitors (deoxyglucose, oligomycin, cyanide, and iodoacetate). Phospholipid degradation was assessed by the release of radiolabeled arachidonate from membrane phospholipids. In this model, the current study demonstrates that (a) cultured myocardial cells display a time-dependent progression of cell injury during ATP depletion; (b) the morphologic patterns of mild and severe cell injury in the cultured cells are similar to those found in intact ischemic canine myocardial models; (c) cultured myocardial cells release arachidonate from membrane phospholipids during ATP depletion; and (d) using two separate combinations of metabolic inhibitors, there is a correlation between the release of arachidonate, the development of severe cellular and sarcolemmal damage, the release of creatine kinase into the extracellular medium, and the loss of the ability of the myocardial cells to regenerate ATP when the metabolic inhibitors are removed. Thus, the present results suggest that during ATP depletion, in cultured neonatal rat myocardial cells, the release of arachidonate from myocardial membrane phospholipids is linked to the development of membrane defects and the associated loss of cell viability.

Topics: Adenosine Triphosphate; Animals; Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Coronary Disease; Creatine Kinase; Cyanides; Deoxyglucose; Iodoacetates; Iodoacetic Acid; Kinetics; Membrane Lipids; Microscopy, Electron; Myocardium; Oligomycins; Phospholipids; Rats

Ischemic myocardium was produced by occluding the left circumflex coronary artery in anesthetized dogs for 10 or 20 min. Autolyzed myocardium was produced by incubating transmural samples of canine left ventricle at 37 degrees C for 5, 10, 15, 20, 40, or 60 min. Tissue pH was recorded continuously in each model using a microcombination pH electrode impaled into the midmyocardium. Mitochondria isolated from both ischemic and autolyzed tissue exhibited marked parallel depressions of oligomycin-sensitive ATPase activity, Km ATP, and Vmax. All of these parameters dropped more markedly during the zero flow autolytic process than during the low flow ischemia characteristic of the canine left circumflex occlusion model. The changes in the ATPase kinetic parameters paralleled closely the drop in tissue pH in each model. These ATPase kinetic changes were then reproduced in vitro both quantitatively and qualitatively by incubating isolated control mitochondria at the same pH values under nonenergizing conditions. It thus became evident that we had, in effect, utilized the oligomycin-sensitive ATPase as an in situ indicator of cell acidosis. Reperfusion of 15-min ischemic myocardium was accompanied by a complete reversal of the acidosis and of the ATPase activity inhibition. The ATPase inhibition demonstrable in vitro in isolated mitochondria occurred when the pH was lowered, but only when there was a concomitant dissipation of the transmembrane electrochemical gradient. The ATPase inhibition was then reversed completely during a subsequent state 4 incubation by a carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive process.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Coronary Disease; Dogs; Female; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Male; Mitochondria, Heart; Oligomycins

Ischemic myocardium was produced by occluding the left circumflex coronary artery in anesthetized dogs. Autolyzed myocardium was produced by incubating transmural samples of canine left ventricle at 37 degrees C. Tissue pH was recorded continuously in each model using a microcombination pH electrode impaled into the midmyocardium. The activities of the five mitochondrial inner membrane enzyme complexes of electron transport and coupled oxidative phosphorylation were assayed as a function of time of ischemia or autolysis. While the activities of complex II (succinate-CoQ reductase) and IV (cytochrome c oxidase) were completely stable, that of complex I (NADH-CoQ reductase) decreased markedly, but largely only after 20 min of ischemia or autolysis. At 20 min and beyond, the decrease in the activity of complex I paralleled closely the decrease in whole mitochondrial oxygen uptake with NAD-linked substrates in both models. The activity of complex III (CoQH2-c reductase) decreased at a more gradual rate during ischemia or autolysis, and its rate of decrease paralleled that of succinate-supported oxygen uptake. The activity of complex V (oligomycin-sensitive ATPase) decreased most rapidly (by 40% in only 5 min of autolysis) but nearly leveled off beyond 20 min in the two models. A strikingly similar pattern of differential enzyme lability was observed in isolated control mitochondria incubated at lowered pH values. The results demonstrate 1) differential enzyme lability within the mitochondrial inner membrane, 2) a connection between severity of acidosis and the degree of enzyme activity loss, and 3) the usefulness of simple tissue autolysis as an analogue of in situ myocardial ischemia.

Topics: Adenosine Triphosphatases; Animals; Carrier Proteins; Coronary Circulation; Coronary Disease; Dogs; Electron Transport; Electron Transport Complex II; Electron Transport Complex III; Electron Transport Complex IV; Female; Hydrogen-Ion Concentration; Kinetics; Male; Membrane Proteins; Mitochondria, Heart; Mitochondrial Proton-Translocating ATPases; Multienzyme Complexes; NAD(P)H Dehydrogenase (Quinone); NADH, NADPH Oxidoreductases; Oligomycins; Oxidoreductases; Oxygen Consumption; Quinone Reductases; Succinate Dehydrogenase

Regional myocardial ischemia was produced in anesthetized pigs by occluding the left anterior descending coronary artery. Mitochondria were prepared from both normally perfused and ischemic myocardium after 2 h of occlusion. Mitochondria from the ischemic area exhibited an 89% increase in cholesterol content from 32.7 +/- 1.9 (control) to 62.0 +/- 0.47 (ischemic) nmol/mg protein with no change in either total phospholipid content or in membrane fatty acid composition. This increase in mitochondrial membrane cholesterol was accompanied by an increase in membrane microviscosity as indicated by increased fluorescence polarization using the fluorescent membrane probe, 1,6-diphenyl-1,3,5-hexatriene. In these same experiments the Arrhenius plot discontinuity temperature of oligomycin-sensitive adenosinetriphosphatase (ATPase) activity fell from 20.0 to 14.2 degrees C. Our results suggest that, during the myocardial ischemic process in pigs, there is an intracellular redistribution of free cholesterol that produces a marked increase in mitochondrial membrane cholesterol content. This appears to produce an altered mitochondrial membrane lipid bilayer packing, resulting in increased membrane microviscosity and, possibly, altered inner membrane ATPase function. Intracellular cholesterol redistribution may thus contribute to the cell membrane damage that occurs during the myocardial ischemic process.

Topics: Adenosine Triphosphatases; Animals; Cholesterol; Coronary Disease; In Vitro Techniques; Membrane Fluidity; Microscopy, Fluorescence; Mitochondria, Heart; Oligomycins; Swine

Left anterior descending coronary artery occlusion in anesthetized pigs produced a stable transmural ischemia characterized by a rapid and then sustained loss of blood flow and mechanical function. After 2 h of occlusion, mitochondria from the ischemic area exhibited a 36 +/- 6% drop in state 3 respiratory activity (QO2) supported by the NAD-linked substrates, glutamate plus malate, but only a 5 +/- 3% decrease in QO2 with succinate plus rotenone. The activity of electron transfer complex I (NADH-CoQ reductase) decreased commensurately by 33 +/- 4% with the decrease in QO2 with NAD-linked substrates. Consistent with the nearly unchanged QO2 with succinate plus rotenone, the activities of electron transfer complexes III and IV decreased only slightly by 9 +/- 5% and 9 +/- 4%, respectively. Mitochondrial ATPase (complex V) activity decreased by 48 +/- 2% with little change in its oligomycin sensitivity. A 48% drop in ATPase activity was shown, by means of oligomycin titrations, to correspond to a 32% decrease in NAD-linked substrate supported QO2. The decreases observed in NADH-CoQ reductase and ATPase activities each account nearly quantitatively for the impaired mitochondrial phosphorylating respiration observed during sustained myocardial ischemia. These results suggest that mitochondrial inner enzyme complexes I and V are important sites of cellular injury in myocardial ischemia.

Topics: Adenosine Triphosphatases; Animals; Coronary Circulation; Coronary Disease; Female; Male; Mitochondria, Heart; Myocardium; NAD(P)H Dehydrogenase (Quinone); NADH, NADPH Oxidoreductases; Oligomycins; Quinone Reductases; Regional Blood Flow; Swine