Metabolism of erucic acid in the isolated perfused rat heart.

In the present work the uptake and utilization of [14C]erucic acid by the perfused rat heart has been investigated and compared with those of [14C]-palmitic acid. Both fatty acids were found to be taken up by the heart at the same rate. On the other hand, the incorporation of erucic acid into tissue lipid during 30 min perfusion were significantly high and CO2 production low as compared with palmitic acid. Incorporation of erucic acid into diacylglycerol, triacylglycerol and cholesterol ester was considerably higher than that of palmitic acid. During a 30-min period, a large amount of [14C]erucic acid was accumulated in tissue fatty acid fraction. Similarly, relatively high labelling was found in the fatty acid and diacylglycerol fraction during the initial 300 s of perfusion with erucic acid. When [14C]erucic acid and unlabelled palmitic acid was used, the radioactivity was very high in the fatty acid fraction of the heart lipid in comparison with the experiment when [14C]palmitate and unlabelled erucic acid was used. Therefore, erucic acid is poorly oxidized by the heart and is preferentially incorporated into heart lipids. There was relatively high incorporation of [14C]erucic acid into diacylglycerol and addition of unlabelled palmitic acid tended to decrease it, probably converting more diacylglycerol to triacylglycerol. When [14C]palmitic acid and erucic acid were used together, incorporation to triacylglycerol was high and diacylglycerol low. These results, therefore suggest that palmitic acid is a more suitable acyl donor than erucic acid for the C-3 position of triacylglycerol, especially when the diacylglycerol contains erucoyl moieties.

Positional and fatty acid specificity of monoacyl- and diacylglycerol 3-phosphate formation by rabbit heart microsomes.

Fatty acid selectivity of acyl-CoA:glycerol-3-phosphate acyltransferase (EC 2.3.1.15) and acyl-CoA:monoacylglycerol-3-phosphate acyltransferase (EC 2.3.1.52) of the microsomal fraction prepared from rabbit heart was studied. 1. The rate of acylation of glycerol 3-phosphate was increased proportionally with the concentration of acyl-CoA. The maximum rate was reached at 0.3 mumol acyl-CoA per ml. Palmitoyl-, oleoyl- and linoleoyl-CoA all served equally well as acyl donors, and produced approximately equal amounts of mono- and diacylglycerol 3-phosphate. The rate of reaction measured in the presence of two acyl-CoA esters was similar to that in the presence of a single acyl-CoA. 2. Treatment of synthesized monoacylglycerol 3-phosphate with Crotalus adamanteus venom phospholipase A2 (EC 3.1.1.4) and that with phosphatidate phosphatase (EC 3.1.3.4) indicated that the heart enzyme synthesized almost exclusively 1-acylglycerol 3-phosphate regardless of the kind of acyl donor. 3. Hydrolysis of diacylglycerol 3-phosphate with phospholipase A2 revealed that there was a slight preference for linoleoyl-CoA at position 2 and a slight discrimination against palmitoyl- and stearoyl-CoA at position 2, when diacylglycerol 3-phosphate was synthesized in the presence of a mixture of acyl-CoA esters. When diacylglycerol 3-phosphate was formed in the presence of a single acyl-CoA, the fatty acid distribution was random. 4. The rate of acylation of 1-palmitoylglycerol 3-phosphate by rabbit heart microsomal fraction was increased proportionally to the increasing concentrations of 1-palmitoylglycerol 3-phosphate up to 50 nmol per ml; higher concentrations were inhibitory. Differences in the activities measured with palmitoyl-, oleoyl- and linoleoyl-CoA as acyl donors were negligible. When stearoyl-, arachidonoyl- and erucoyl-CoA acted as acyl donors, the rates of reaction were low. 5. The acyl-CoA:1-palmitoylglycerol-3-phosphate acyltransferase activity increased proportionally to the increasing concentrations of acyl-CoA up to 10 nmol per ml; acyl donor specificity was similar to that found above [4]. The acyltransferase showed

Mitochondrial dysfunction observed in situ in cardiomyocytes of rats in experimental diabetes.

OBJECTIVE: The aim was to investigate effects of experimental diabetes and insulin treatment on heart myocytes, particularly on the mitochondrial function studied in situ in isolated cardiomyocytes. METHODS: 20 male Sprague-Dawley rats (140-160 g) were made diabetic by intraperitoneal streptozotocin, 70 mg.kg-1. Ten then received daily subcutaneous injections of ultra lente insulin (starting dose of 3 units.d-1) for 7-15 d from the 20th day after streptozotocin. There was a control group of 11 rats. The rats were killed 21-35 d after the induction of diabetes, and heart myocytes were isolated by collagenase digestion. The 45[Ca]2+ uptake of mitochondria in situ in permeabilised myocytes, the transmembrane potential gradient of mitochondria, and the respiration of myocytes, as well as the cell yield and cell [45Ca]2+ uptake, were examined. RESULTS: Mitochondrial uptake of [45Ca]2+ was significantly decreased in the diabetic group compared to control at cytosolic calcium concentrations between 760 nM and 44.6 microM. The mitochondrial potential of diabetic myocytes, estimated from the distribution of [3H]triphenylmethylphosphonium+, was slightly but significantly decreased from the control value. Cell respiration, measured polarographically in the presence of pyruvate and malate or succinate as oxidisable substrates, and with or without 2,4-dinitrophenol, was decreased by diabetes. The rapidly exchangeable [45Ca]2+ content in the myocyte with intact sarcolemmal membrane ("cell Ca2+ uptake") and the yield of cells from heart tissue were also diminished in diabetic rats. These changes were returned to normal by insulin treatment of 7 d or longer. CONCLUSIONS: Insulin deficiency at early stages causes defects of mitochondrial function detectable in situ in cardiomyocytes. This suggests the possibility that such alterations are causative factors in the development of diabetic cardiomyopathy.

Membrane phospholipids and plasmalogens in the ischemic myocardium.

This review begins with discussion concerning the effects of changes in phospholipid compositions on membrane functions. Next, pathogenetic mechanisms of ischemic cell damage are reviewed; a) membrane phospholipid breakdown, caused by the activation of phospholipases, as well as the intracellular Ca2+ overload, and b) univalent oxygen reduction and lipid peroxidation, probably all play important roles. Consequently, hydrophilic metabolites of lipid peroxidation may accumulate in the hydrophobic membrane bilayer during ischemia, causing membrane dysfunction. Free radicals may involve the cross-linking of membrane proteins as well. Previous results in support of the free radical hypothesis of myocardial ischemic injury are described; they were obtained from ESR studies, measurements of tissue antioxidants, determinations of lipid breakdown products, and studies using scavengers. The distribution, biosynthesis and physical-chemistry of plasmalogens are then discussed. Excepting the platelet activating factor recently discovered, only fragmentary information is available concerning the function of plasmalogens. It is possible that membrane plasmalogens, which contain large amounts of polyunsaturated fatty acids, are vulnerable to free radical/ischemic injury.

Oxidant injury to isolated heart cells.

Recent evidence suggests that free radicals are generated in the heart during the reperfusion which follows ischemia. Intracellular accumulation of calcium has been postulated to be an important pathogenic factor in a number of disease states, including reperfusion injury. Therefore, in this study, the effects of various oxidants on calcium uptake by isolated rat heart cells were investigated. Ammonium persulphate, t-butyl hydroperoxide and phenazine methosulphate increased the number of cells in contracture in both a concentration dependent and time dependent manner, while 45Ca content of cardiomyocytes was decreased by oxidant in proportion to its concentration. Carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) dependent (mitochondrial) and CCCP independent (sarcoplasmic reticulum) 45Ca contents in chemically skinned myocytes were reduced by the oxidants. By contrast, hydrogen peroxide raised 45Ca content of cardiomyocytes and did not reduce sarcoplasmic reticulum 45Ca content, although mitochondrial 45Ca content was decreased. Release of 45Ca from mitochondria and sarcoplasmic reticulum in saponin treated myocytes was accelerated by hypochlorous acid and hydrogen peroxide. The authors conclude that oxidants other than hydrogen peroxide inhibited intracellular uptake of calcium and accelerated calcium release, thus raising the cytosolic calcium concentration and causing cell contracture. The net influx of calcium across sarcolemmal membrane was decreased by these oxidants.

Effect of bovine serum albumin on monoacyl- and diacylglycerol 3-phosphate formation in mitochondrial and microsomal fractions of rabbit hearts.

The formation of monoacyl- and diacylglycerol 3-phosphate (P) by rabbit heart mitochondrial and microsomal fractions was studied by varying the concentration of acyl-CoA and that of bovine serum albumin in the assay system. The two subcellular fractions were prepared by the conventional differential centrifugation technique. The optimal concentration of acyl-CoA for both mitochondrial and microsomal acylation of glycerol 3-P was shifted to a higher range of acyl-CoA concentrations by greater amounts of albumin. A similar shift in the acyl-CoA concentration-enzyme activity relationship was observed in the acylation reaction of 1-palmitoylglycerol 3-P by the heart microsomes. The addition of albumin increased slightly the rate of diacylglycerol 3-P accumulation but increased greatly the rate of monoacylglycerol 3-P accumulation at any concentration of acyl-CoA; the effect was observed with mitochondrial or microsomal fraction as the crude enzyme source. Moreover, palmitoyl-CoA and linoleoyl-CoA served equally well as the acyl donor for the acylation reaction. However, relatively more monoacyl- than diacylglycerol 3-P was accumulated in the assays with rabbit heart mitochondrial fraction in the presence of albumin, whereas more diacyl- than monoacylglycerol 3-P was formed by the microsomal fraction. As a result, the microsomal diacyl:monoacyl-glycerol 3-P ratio was invariably greater than the mitochondrial ratio at a given concentration of acyl-CoA and albumin.

Effects of leukocyte-derived oxidants on sarcolemmal Na,K,ATP-ase and calcium transport.

Our study demonstrated that the Na,K,ATPase activity and ouabain binding sites were reduced by oxidants. Sarcolemmal calcium transport was also inhibited by hydrogen peroxide and HOC1. The action of HOC1 on the sarcolemmal functions was 2-3 orders of magnitude more powerful than that of hydrogen peroxide. Effects of hydrogen peroxide consisted of two components, i.e., the first, highly sensitive one, most probably mediated by Fe-catalyzed, site-specific free radical formation, and the second, less potent action by (high concentrations of) hydrogen peroxide. Finally, very low concentrations of hydrogen peroxide potentiated Na,K,ATPase activities when assayed using myocytes.

Free radical effects on membrane protein in myocardial ischemia/reperfusion injury.

Free radical generation may be the principal pathogenic factor responsible for the initiation of ischemia/reperfusion myocardial cell damage. Circumstantial evidence is in favour of the view that impaired function of the membraneous components of ischemic cells is associated with oxygen radical-induced alterations in proteins and lipids. Thus, lipid antioxidants and free radical scavengers as well as antagonists of protein modification may all be required for therapeutic intervention of this aspect of ischemia/reperfusion injury.

Effects of hydrogen peroxide on mitochondrial enzyme function studied in situ in rat heart myocytes.

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.

The effects of myocardial ischemia and nisoldipine pretreatment on the asymmetric distribution of phosphatidylethanolamine in a canine heart sarcolemmal preparation.

We examined the distribution of phosphatidylethanolamine (PE) in the membrane bilayer of sarcolemmal preparation isolated from the ischemic and nonischemic areas of dog ventricles. The membrane preparation, isolated by the Reeves and Sutko's method, was purified ninefold over homogenates as judged from the results of measurements of (Na+K+)-ATPase and K+-p-nitrophenylphosphatase activities, sialic acid, and cholesterol. Sealed vesicles were comprised of 60% inside-out-oriented and 40% rightside-out-oriented vesicles; 30% of the total were unsealed vesicles. The results obtained from the incubation of the membrane preparation with 2,4,6-trinitrobenzenesulfonic acid (TNBS) and cycloheptaamylose-fluorescamine complex, both of which served as nonpermeable chemical probes, indicated that 80% of the total PE was accessible from the outside. By contrast, it was possible to label up to 98% of the PE by using a permeable probe, 1-fluoro-2,4-dinitrobenzene. These results suggest that PE is predominantly localized in the cytosolic side of the sarcolemmal membrane bilayer in the dog heart. Ischemic lesion was produced in the dog heart by the occlusion of a branch of the left anterior descending coronary artery for 1.5 hr followed by 3 hr of reflow. The concentrations of both total phospholipid and phosphatidylcholine and PE in the sarcolemmal fraction prepared from the ischemic area of the myocardium were significantly decreased as compared to those from the nonischemic area. The magnitude of labeling sarcolemmal PE by TNBS was reduced in the preparation from the ischemic area as compared to that from the nonischemic area. This difference was abolished when the dog received nisoldipine (an iv injection of 5 micrograms/kg twice) or chlorpromazine (infusion at a rate of 10 micrograms/kg X min plus an iv injection of 400 micrograms/kg twice). These results suggest that ischemia decreased primarily the membrane PE existing at the cytosolic side of the sarcolemmal membrane and that pharmacological intervention can prevent the change in membrane lipids induced by ischemia.

Orientation of vesicles isolated from baso-lateral membranes of renal cortex.

Baso-lateral membranes were isolated from the canine and porcine kidney cortex by several different methods currently in use. Sidedness of the isolated membrane vesicles was determined by procedures using 1. ouabain-sensitive (Na+K+)ATPase assays in the presence and in the absence of sodium dodecylsulfate or digitoxigenin plus monensin, 2. (Na+, K+, Mg2+)ATPase assays with valinomycin, 3. sialidase accessibility, and 4. binding of hydrophilic and lipophilic cardiac glycosides. The (Na+K+)ATPase activity in the membrane preparation was increased 10-fold of that found in the crude homogenate. Isolated membrane vesicles, prepared by different techniques, were all found to be overwhelmingly of right-side-out orientation;namely, right-side-out = 51-68%, inside-out = 4-13%, and unsealed vesicles = 26-42%. Results of sidedness determinations by different methods showed a good agreement. Thus, predominantly right-side-out oriented vesicles are formed during conventional isolation procedures for membranes of the kidney cortex.

Effects of N-(2-mercaptopropionyl)glycine on ischemic-reperfused dog kidney in vivo and membrane preparation in vitro.

The results of our experiments demonstrated that one hour of ischemia followed by one hour of reflow in the kidney caused a reduction in (Na+K+)ATPase activity and microsomal sulfhydryl content as well as an increase in microsomal lipid peroxidation. Renal venous malondialdehyde concentration was increased soon after reperfusion of the ischemic kidney. All these changes were rectified by an infusion of 0.123 mmol N-(2-mercaptopropionyl)glycine/kg over a 70 min period. On the other hand, an in vitro addition of 0.01-0.5 mM N-(2-mercaptopropionyl)glycine to a membrane preparation in the presence of H2O2 and Fe3+ did not prevent but rather potentiated the free radical effect on the enzyme activity. However, addition of superoxide dismutase alone or with catalase together with 2-MPG were effective in preventing the enzyme depression induced by H2O2. The results therefore indicate that free radical generation participates in the evolution of ischemia/reperfusion cell injury and thiol-reducing agents may be beneficial in alleviating the cell damage in vivo.

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.

Na+/Ca2+ exchange of isolated sarcolemmal membrane: effects of insulin, oxidants and insulin deficiency.

In this study we prepared sarcolemmal fractions from bovine and rat hearts; their Na+K+ ATPase activities, measured in the presence of saponin to unmask latent Na+K+ ATPase, were 59.4 and 48.8 mu mol Pi/mg protein.h, respectively. The rate of Na+ dependent Ca2+ uptake was linear for the first 10 s and a plateau was reached in 3 min. Oxidation by free radical generation either with H2O2, FeSO4 plus DTT or xanthine oxidase plus hypoxanthine stimulated Na+/Ca2+ exchange in a time-dependent manner. The stimulation was abolished by deferoxamine or o-phenanthroline. By contrast, oxidation by HOCl inhibited Na+/Ca2+ exchange in proportion to its concentration, and this inhibition was antagonized by DTT. DTT alone had no effect on the exchange. Insulin stimulated Na+/Ca2+ exchange, its maximal effect was attained after 30 min incubation with 100 mu units/ml. N-ethylmaleimide inhibited the exchange both in the presence and in the absence of insulin. Sarcolemmal fractions prepared from hearts of alloxan-treated, acutely diabetic rats showed a significant decrease in Na+/Ca2+ exchange. Addition of insulin in vitro significantly stimulated Na+/Ca2+ exchange of both diabetic and control groups. The results indicate that sarcolemmal Na+/Ca2+ exchange function is modulated by oxidation-reduction states and by the presence of insulin.

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.

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.