Our previous work indicated that energy transduction, as measured by myocyte respiration, was inhibited by hydrogen peroxide, but the mitochondrial membrane potential was relatively unaffected. Therefore, we determined in the present study the critical steps in mitochondrial energy transduction by measuring the sensitivity to hydrogen peroxide of NADH-CoQ reductase, ATP synthase, and adenine nucleotide translocase in situ in myocytes. Adult rat heart cells were isolated using collagenase and incubated in the presence of 0.1-10 mM hydrogen peroxide for 30 min. Activities of NADH-CoQ reductase and oligomycin-sensitive ATP synthase were assayed enzymatically with sonicated myocytes, and adenine nucleotide translocase activities were determined by atractyloside-inhibitable [14C]ADP uptake of myocytes, permeabilized by saponin. The NADH-CoQ reductase and ATP synthase activities were inhibited to 77% and 67% of control, respectively, following an exposure to 10 mM hydrogen peroxide for 30 min. The adenine nucleotide translocase activities were inhibited in a concentration- and time-dependent manner and by 10 mM hydrogen peroxide to 44% of control. The dose-response relationship indicated that the translocase was the most susceptible to hydrogen peroxide among the three enzymes studied. Combined treatment of myocytes with 3-amino-1,2,4-triazole, 1,3-bis(2-chloroethyl)-1-nitrosourea and diethyl maleate (to inactivate catalase, to inhibit glutathione reductase activity, and to deplete glutathione, respectively) enhanced the sensitivity of translocase to hydrogen peroxide, supporting the view that the cellular defense mechanism is a significant factor in determining the toxicity of hydrogen peroxide. The results indicate that hydrogen peroxide can cause dysfunction in mitochondrial energy transduction, principally as the result of inhibition of adenine nucleotide translocase.

Adenosine Diphosphate↗

PubMedDate not supplied

Role of cellular defense against hydrogen peroxide-induced inhibition of myocyte respiration.

Hydrogen peroxide (H2O2) serves as a precursor for highly reactive oxygen intermediates. However, the respiratory function of myocytes is relatively resistant to exogenously administered H2O2. In this study, we examined whether or not the reduction of cellular defense increases the toxicity of H2O2. Rat heart myocytes were isolated by collagenase digestion. Respiratory rates of myocytes, suspended in a medium containing sucrose, 3-N-morpholino-propanesulfonic acid, EGTA and bovine serum albumin, were determined polarographically in the presence of pyruvate and malate with or without 2,4-dinitrophenol (DNP). Mitochondrial membrane potentials were measured by using [3H]triphenylmethylphosphonium+. Cellular defense was attenuated by i) inhibiting the catalase activity by 3-amino-1,2,4-triazole (AT), ii) reducing the glutathione concentration by diethyl maleate (DEM) or ethacrinic acid (EA), and iii) permeabilizing the sarcolemmal membrane by saponin. The dose-response relationship between H2O2 (0.1-5 mM) and mitochondrial membrane potential was not greatly affected by these experimental conditions. Myocyte respiration was inhibited by 5 mM H2O2, particularly that measured in the presence of DNP (48% of control). DEM treatment did not significantly affect the respiratory inhibition by H2O2, whereas the degree of inhibition was somewhat greater following EA or AT treatment. By contrast, the sensitivity of cellular respiration to H2O2 was potentiated approximately two orders of magnitude by the permeabilization of sarcolemmal membrane; thus, 100 microM H2O2 inhibited both DNP-stimulated and unstimulated respiration to 17% and 35% of control, respectively. The results indicate that factors existing in the sarcolemma and/or in the cytosol, which become ineffective and/or are diluted, respectively, following permeabilization with saponin, are important cellular defense mechanisms in alleviating the toxic effect of exogenous H2O2 on the respiration of mitochondria in situ in myocytes.

Amitrole↗

PubMedDate not supplied

Myocardial nucleic acid synthesis following constriction of the aorta in rats.

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Adenine↗

PubMedDate not supplied

Effect of oxidants on Na,K,ATPase and its reversal.

Ischemia-reperfusion heart cell injury may be mediated, at least in part, through the generation of oxy radicals. Therefore, mechanisms of action of two oxidants on a membrane model, partially purified Na,K,ATPase, were investigated. Effects of H2O2, an oxygen intermediate postulated to play a primary role in reperfusion injury, on the function of the enzyme were time-dependent and potentiated by Fe ions. The inhibition of enzyme activity was prevented by chelators, but not by hydroxyl radical scavengers. The results support the view that the possible mode of enzyme modification involves H2O2-derived, Fe ion-catalyzed, localized ("site-specific") hydroxyl radical formation. The action of hypochlorous acid (HOCl), a powerful oxidant postulated to be produced by activated neutrophils, was quantitatively similar to that of H2O2 plus Fe ions in causing enzyme dysfunction. This is partly because relatively large doses of oxidants were required, due to the presence of physiological anti-oxidant defense mechanisms in the membrane. Although a combination of deferoxamine (Fe ion chelator) and dithiothreitol (DTT) (sulfhydryl reducing agent) was most effective in preventing the enzyme modification, once enzyme inactivation by oxidants is in progress, deferoxamine plus DTT could only arrest further deterioration of the enzyme function. Therefore, the oxidant-induced change in membrane dysfunction advances with time; the advance can be stalled, but the enzyme activity cannot be restored to normal.

Adenosine Diphosphate↗

PubMedDate not supplied

The effect of a calcium channel antagonist, Nisoldipine, on the ischemia-induced change of canine sarcolemmal membrane.

Ischemic injury was produced in the dog heart by occluding the left anterior descending coronary artery just below the second diagonal branch for a duration of 1.5 h followed by the release of occlusion. Nisoldipine, 3.5 micrograms/kg was injected intravenously 10 min before the occlusion and again 10 min before the commencement of reperfusion. The activity of serum creatine phosphokinase greatly increased after the reperfusion, and this increase was significantly suppressed by Nisoldipine. This drug, in addition, prevented ischemia-induced myocardial hemorrhage and premature ventricular contraction. Sarcolemmal membrane vesicles were prepared from an ischemic and non-ischemic portions of the myocardium 3 h after the commencement of reflow. The fraction was purified approximately 12-fold with respect to ouabain-sensitive (Na+K+)-ATPase as an indicator; contamination of mitochondria was minimum with cytochrome c oxidase as an indicator. Without treatment of Nisoldipine, the total amount of sarcolemmal phospholipid obtained from the ischemic area, as well as the amounts of phosphatidyl-choline and phosphatidyl-ethanolamine, were significantly decreased as compared with those obtained from the non-ischemic area. Nisoldipine treatment abolished the decrease in the sarcolemmal phospholipids, total as well as phosphatidyl-choline and -ethanolamine, induced by ischemia plus reperfusion. Therefore, our work indicates that the Ca++ channel antagonist, Nisoldipine, suppresses the ischemia-induced increase in phospholipid breakdown of cardiac sarcolemma probably through its inhibitory effect on the Ca++-mediated activation of membrane phospholipase, through its vasodilatory action, or both.

Animals↗

PubMedDate not supplied

Sensitivity to oxidants of mitochondrial and sarcoplasmic reticular calcium uptake in saponin-treated cardiac myocytes.

Calcium transport functions of mitochondria and sarcoplasmic reticulum (SR) were studied without prior extraction using isolated rat heart myocytes permeabilized with saponin. Calcium uptake by SR was rapid and its affinity was high in comparison to calcium uptake by mitochondria, which had a higher capacity. The sensitivity of uptake to two oxidants, H2O2 and HOCl (hypochlorous acid), depended on the cytosolic calcium concentration; when this was similar to the concentration in diastole (180 nM), HOCl inhibited calcium uptake by mitochondria and SR, whereas when the calcium concentration was 750 nM, mitochondrial calcium uptake showed relatively high resistance, although SR uptake was still markedly inhibited by HOCl. Calcium uptake of both mitochondria and SR was less sensitive to the action of H2O2 than to HOCl, and the H2O2 effect was less dependent on the cytosolic calcium concentration. Therefore, HOCl, when produced by activated leukocytes and supplied to the heart cells, may seriously impair the excitation-contraction coupling function of SR, whereas H2O2, possibly generated directly by mitochondria or generated from superoxide anions, may be tolerated relatively well by heart SR and mitochondria.

Animals↗

PubMedDate not supplied

Effects of chloroquine and Nifedipine on the phospholipid content and enzyme activity in the subcellular fraction of ischemic rat liver.

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Animals↗

PubMedDate not supplied

Association of liposomes with the isolated perfused rabbit heart.

Liposomes were prepared from a mixture of either phosphatidylcholine (PC) and cholesterol (Ch) (7:2) (neutral), PC, dicetylphosphate and Ch (4:1:3) (negative), or PC, stearylamine and Ch (4:1:3) (positive). In addition, as the lipid phase-liposomal marker, 3H- or 14C-Ch was added. Alternatively, 14C-labelled mannitol, glucose or inulin was used as the aqueous phase-marker in some experiments. The liposomes were purified by Sephadex gel chromatography and by using microfilter. After 4 h of sonication, 95% of the total liposomes were found to be smaller than 1.2 microns. The entrapment volume was calculated to be 0.9-1.2 microliters/mumole of lipid. The ratio of lipid radioactivity and aqueous phase-radioactivity, which were found in a non-filtrable portion of the perfusate, remained constant during a heart-perfusion period of 30 min, indicating that the liposomes were stable during the experimental period. The wash-out experiment indicated that the liposomes were distributed in a space of the perfused heart nearly as large as the mannitol space. The liposomes were rapidly taken up by the heart during perfusion in a Langendorff manner. The positive liposomes were taken up by the perfused nonischemic heart at a greater rate than were the negative liposomes. The results with perfused ischemic hearts were equivocal. The liposomal label was found to be distributed unequally in the subcellular fractions, namely, relatively greater amounts (per mg protein) of liposome-bound radioactivity of Ch or mannitol were found in the microsomal fraction than in the mitochondrial or cytosolic fractions of the perfused heart. This distribution pattern was not influenced by the electrical charge of liposomes or by the oxygenation state of the heart perfusion. The accumulation of the liposomal label in the microsomal fraction found in the heart perfusion experiment could not be observed in the experiment in which tissue slices were incubated in the presence of liposomes, or in the experiment in which free mannitol was administered in the heart perfusion.

Animals↗

PubMedDate not supplied

Metabolism of individual fatty acids in heart muscle.

We have studied the incorporation of various long chain fatty acids into tissue lipids using various heart preparations. We found that individual fatty acids are incorporated into various lipid fractions at unequal rates. In particular, erucic acid was shown to be a poor acyl donor for various acylation reactions. Labeling of triacylglycerol by exogenous fatty acid was found to be extremely rapid in the perfused rat heart and in the in vivo heart.

Animals↗