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K J Kako

Publications and source records attributed to K J Kako.

At least 19 recordsLinked to original sources
PubMed1992

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.

Animals↗
PubMed1991

Effects of hydrogen peroxide and hypochlorite on membrane potential of mitochondria in situ in rat heart cells.

Hydrogen peroxide (H2O2) and hypochlorite (HOCl) cause a variety of cellular dysfunctions. In this study we examined the effects of these agents on the electrical potential gradient across the inner membrane of mitochondria in situ in isolated rat heart myocytes. Myocytes were prepared by collagenase digestion and incubated in the presence of H2O2 or HOCl. Transmembrane electrical gradients were measured by distribution of [3H]triphenylmethylphosphonium+, a lipophilic cation. The particulate fraction was separated from the cytosolic compartment first by permeabilization using digitonin, followed by rapid centrifugal sedimentation through a bromododecane layer. We found that the mitochondrial membrane potential (161 +/- 7 mV, negative inside) was relatively well maintained under oxidant stress, i.e., the potential was decreased only at high concentrations of HOCl and H2O2 and gradually with time. The membrane potential of isolated rat heart mitochondria was affected similarly by H2O2 and HOCl in a concentration- and time-dependent manner. High concentrations of oxidants also reduced the cellular ATP level but did not significantly change the matrix volume. When the extra-mitochondrial free calcium concentration was increased in permeabilized myocytes, the transmembrane potential was decreased proportionally, and this decrease was potentiated further by H2O2. These results support the view that heart mitochondria are equipped with well-developed defense mechanisms against oxidants, but the action of H2O2 on the transmembrane electrical gradient is exacerbated by an increase in cytosolic calcium.

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

Increase vs. decrease of calcium uptake by isolated heart cells induced by H2O2 vs. HOCl.

Adult rat heart myocytes were labeled rapidly with exogenous [45Ca2+]. Addition of 2.5 mM H2O2 to the heart cell suspension raised the content of rapidly exchangeable intracellular Ca2+ twofold, whereas addition of 1-30 mM HOCl decreased the Ca2+ content. The H2O2-induced increase in Ca2+ content was dependent on the medium Na+, pH, and temperature but was not significantly affected by addition of verapamil, diltiazem, amiloride, or 3-aminobenzamide. The [3H]ouabain binding to myocytes was suppressed by H2O2, whereas the Ca2+ efflux from myocytes was not influenced. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, reduced Ca2+ content, implying that the H2O2-induced change in Ca2+ content was not directly related to ATP depletion. On the other hand, the H2O2-induced Ca2+ accumulation in myocytes was prevented by deferoxamine or o-phenanthroline. These results suggest that H2O2 inhibited Na+-K+-ATPase, resulting in an increase in intracellular Na+ concentration and stimulation of sarcolemmal Na+-Ca2+ exchange activity, which caused a transient net Ca2+ influx into myocytes. By contrast, HOCl decreased the Ca2+ content of the rapidly exchangeable pool below control levels and this action of HOCl was antagonized by 1,4-dithiothreitol. HOCl accelerated Ca2+ efflux from myocytes. Ca2+ uptake and Ca2+-ATPase of the isolated sarcoplasmic reticular (SR) fraction were highly sensitive to the action of HOCl. Ca2+ uptake by intracellular sites, studied with myocytes permeabilized with digitonin, was inhibited by both H2O2 and HOCl. Thus these results suggest that HOCl inhibits the SR Ca2+ pump, resulting in the observed acceleration of Ca2+ efflux from and decline in Ca2+ content of myocytes.

Animals↗
PubMed1989

Pharmacological intervention in oxidant-induced calcium pump dysfunction of dog heart.

Micromolar concentrations of HOCl, an oxidant produced by activated neutrophils, inhibited Ca2+ uptake and Ca2+ATPase of isolated dog heart sarcoplasmic reticulum (SR). DTT antagonized completely the HOCl effect only when it was given within 5 min after the addition of HOCl. When the pharmacological intervention was delayed, the recovery with DTT was not complete, and administration of DTT 30 min after the start of HOCl's reaction with SR resulted in only a small improvement in SR Ca2+ uptake. Although H2O2 and Fe ion-chelate (a free radical-generating procedure) also inhibited Ca2+ uptake and ATPase, the concentrations required were very large. The response of cardiac sarcolemmal and skeletal muscle SR calcium pumps to oxidants was similar to that of the cardiac SR calcium pump.

Animals↗
PubMed1989

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.

Animals↗
PubMed1988

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.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

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.

Amino Acids, Sulfur↗
PubMed1987

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.

Animals↗
PubMed1986

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.

Animals↗
PubMed1979

Acylglycerol structure of mustard seed oil and of cardiac lipids of rats during dietary lipidosis.

Stereospecific degradation and combined gas chromatographic--mass spectrometric (gc/ms) analysis were employed in a detailed investigation of the triacylglycerol structure of mustard seed oil and of the triacylglycerols transiently accumulating in the hearts of young rats receiving the oil in their diet. It was shown that feeding of mustard seed oil at 40% of the daily caloric requirement resulted in a deposition of cardiac triacylglycerols containing a high proportion of enantiomers of a positional distribution and molecular association of fatty acids which were closely similar to those found in the dietary oil. Complete structures were derived for a total of 88 species representing 75 to 85% of the triacylglycerols. About 90% of the accumulated triacylglycerol contained at least one long-chain (C20--C22) monounsaturated fatty acid per molecule. The long-chain acids were confined mainly to the primary positions and preferentially to the sn-3-position of the glycerol molecule. The dietary lipidosis, is, therefore, accompanied by little or no accumulation of the normal rat tissue triacylglycerols containing C16 and C18 fatty acids. It is suggested that the deposition and eventual clearance of the enantiomeric long-chain triacylglycerols in the rat heart during mustard seed oil feeding may be largely a result of a gradual change in specificity of the cardiac lipases.

Animals↗
PubMed1977

Phosphatidic acid synthesis in the heart. 1. Effect of age and species difference in the mitochondrial and microsomal synthesis.

Rates of syntheses of monoacyl- and diacyl-glycerol 3-P (phosphate) were determined in the mitochondrial and microsomal fractions prepared from hearts of rats and rabbits, to compare characteristics of the acylation reactions by the two subcellular fractions. The assays were carried out with the subcellular fractions prepared from (i) hearts of hyperthyroid animals, and (ii) hearts of newborn and weanling rats. In addition, the effect of an addition of bovine serum albumin in the assay system was examined. (1) Administration of thyroid hormones increased the acyltransferase activity in rabbit hearts but not that in rat hearts. (2) Mitochondrial and microsomal fractions of hearts of newborn rats acylated glycerol 3-P at a rate 1.3-4 times greater than those of adult rats. The rate of acylation by the mitochondrial fraction of weanling rats was also high, but the rate of microsomal acylation was slightly lower than that of adult rats. By contrast, in newborn rats, diacylglycerol 3-P formation by the liver microsomes was not greater than that of the adults, although its formation by the newborn liver mitochondria was greater. (3) The accumulation of monoacylglycerol 3-P during the assay was accelerated by the addition of increasing amounts of bovine serum albumin. Therefore, the monoacylglycerol 3-P formation was less than 10% of the diacylglycerol 3-P formation in the assay containing no albumin and with the rat subcellular fractions, whereas nearly four times more monoacyl- than diacyl-glycerol 3-P was synthesized in the presence of 20 mg albumin. (4) The ratio of monoacyl- to diacyl-glycerol 3-P formation by the mitochondrial fraction was greater than that of microsomal fraction at any concentration of albumin in both rats and rabbits. At an equal albumin concentration in the assay, relatively more diacyl- than monoacyl-glycerol 3-P was formed in the mitochondrial fraction of newborn rat hearts as compared with adult hearts; (5) In conclusion, our data concerning the age and species differences in acyltransferase activities support a view that the mitochondrial fraction of both rat and rabbit hearts, in addition to the microsomal enzymes, is capable of catalyzing the de novo synthesis of phosphatidic acid.

Acyltransferases↗