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K Takeshige

Publications and source records attributed to K Takeshige.

At least 91 records · Page 5Linked to original sources
PubMed1989

Oxidative damage in selenium deficient hearts on perfusion with adriamycin: protective role of glutathione peroxidase system.

The protective effects of the glutathione peroxidase system against functional damage induced by perfusion of isolated hearts with adriamycin, an anthracycline antibiotic, were studied. We used selenium deficient rats, in which cardiac glutathione peroxidase activity was only 3% of control rats. Both contractile tension and coronary flow decreased during perfusion with the antibiotic. The degree of decline was significantly greater in the selenium deficient hearts than in the control hearts. The increase in malondialdehyde, a product of lipid peroxidation, induced by adriamycin perfusion was more evident in selenium deficient hearts, though the level of reduced glutathione was well maintained. Isolated mitochondrial function also decreased after aerobic adriamycin perfusion and the decrease was greater in selenium deficient rats. These observations indirectly suggest that the decrease in cardiac function induced by adriamycin is protected by the glutathione peroxidase system and that the decrease may be due, at least in part, to damage to the mitochondria caused by oxygen radicals generated by adriamycin.

Animals↗
PubMed1989

Effect of 2',3'-dialdehyde NADPH on activation of superoxide-producing NADPH oxidase in a cell-free system of pig neutrophils.

We studied the effect of the 2',3'-dialdehyde derivative of NADPH on the activation of superoxide-producing oxidase in a cell-free system of pig neutrophils. The system consisted of a membrane fraction, two cytosolic fractions prepared by gel filtration, and arachidonic acid. Preincubation of one of the cytosolic fractions with the derivatives of NADPH and NADP+ caused the loss of its ability to activate the enzyme. The inactivation was effectively prevented by the addition of NADPH and NADP+. Neither the membrane fraction nor the other cytosolic fraction was affected by the derivatives. The results indicate that the NADPH binding component of the oxidase is present in the cytosolic fraction and may be translocated to the membrane fraction during the activation process in the cell-free system.

Animals↗
PubMed1989

Measurement of the Cytoplasmic and Vacuolar Buffer Capacities in Chara corallina.

The cytoplasm and the vacuole were isolated from internodal cells of Chara corallina by using the intracellular perfusion technique, and their buffer capacities (beta(i)) were determined from the titration curves. The pH of the isolated vacuolar sap was 5.19 +/- 0.029 (mean +/- standard error). At this pH, beta(i) was minimal and amounted to 0.933 +/- 0.11 millimoles H(+)/pH unit/liter vacuolar sap. The pH of isolated cytoplasm was 7.22 +/- 0.028. beta(i) was minimal in this pH region and amounted to 14.2 +/- 0.80 millimoles H(+)/pH unit/liter cytoplasm. When 1% (volume/volume) Triton X-100 was added to the cytoplasmic solution to permeabilize the subcellular organelles, the cytoplasmic pH increased to 7.32 +/- 0.026, where beta(i) was 20.35 +/- 2.66 millimoles H(+)/pH unit/liter cytoplasm. This shows that alkaline subcellular compartments exist in the cytoplasm and also that the cytoplasmic pH before adding Triton X-100 may represent the cytosolic pH. These data indicate that the pH values of the cytoplasm and the vacuole are regulated at the values where the beta(i) values are minimal. This suggests that ATP- and inorganic pyrophosphate-dependent H(+) pumps in the plasma membrane and the tonoplast could efficiently regulate the pH of both cytoplasm and vacuole in Chara internodal cells.

Journal Article↗
PubMed1989

Salt Stress-Induced Cytoplasmic Acidification and Vacuolar Alkalization in Nitellopsis obtusa Cells : In VivoP-Nuclear Magnetic Resonance Study.

Time courses of cytoplasmic and vacuolar pH changes under salt stress were monitored by in vivo(31)P-nuclear magnetic resonance spectroscopy in intact cells of Nitellopsis obtusa. When cells were treated with 100 millimolar NaCl for 2 hours, the cytoplasmic pH deceased from 7.2 to 7.0, while the vacuolar pH increased from 4.9 to 5.2. This salt-induced breakdown of the pH gradient between the cytoplasm and the vacuole was also confirmed through direct measurements of change in vacuolar pH with a micro-pH electrode. We speculate that the intracellular pH changes induced by the salt stress mainly results from the inhibition of the H(+)-translocating pyrophosphatase in the vacuolar membrane, since this H(+)-translocating system is sensitive to salt-induced increase in the cytoplasmic [Na(+)] and a simultaneous decrease in the cytoplasmic [K(+)]. Since disturbance of the cytoplasmic pH value should have serious consequences on the homeostasis of living cells, we propose that the salt-induced intracellular pH changes are one of initial and important steps that lead to cell death.

Journal Article↗
PubMed1989

Metabolism of stimulated polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNL) release superoxide anions formed by a membrane-bound NADPH oxidase induced by stimulations. Properties of the inducers and their antagonists indicate that Ca2+, GTP-binding protein (G-protein), phospholipase C and Ca2+, phospholipid-dependent protein kinase (C-kinase) are mainly associated with the stimulation of receptors. Low concentrations of ATP induce the oxidase accompanied by the increase in the intracellular Ca2+ due to the flux from the medium and the storage site. ATP-gamma-S, UTP and ITP are effective but mononucleotides, dinucleotides, GTP and CTP are not. Leukotriene B4 (LTB4) which acts as a chemotactic agent and the inducer of the NADPH oxidase is catabolized. It is hydroxylated by a specific cytochrome P450 and then oxidized to a carboxy derivative by a cytosolic alcohol dehydrogenase and a microsomal aldehyde dehydrogenase in PMNL. Active NADPH oxidase was obtained by incubating membrane and cytosolic components of resting PMNL in the presence of sodium dodecyl sulfate (SDS). Two cytosolic components were obtained by an affinity chromatography on 2',5'-ADP Sepharose. One component is active in the presence of GTP or GTP-gamma-S and the other component in the presence of another cytosolic fraction.

Adenosine Triphosphate↗
PubMed1988

NAD+-dependent oxidation of 20-hydroxyleukotriene B4 to 20-carboxyleukotriene B4 by rat liver cytosol.

20-Hydroxyleukotriene B4 was converted by rat liver homogenates in the presence of NAD+ to a more polar product on reverse-phase high-performance liquid chromatography. The product was identified as 20-carboxyleukotriene B4 by straight-phase high performance liquid chromatography, ultraviolet spectrophotometry and gas chromatography-mass spectrometry. The oxidative activity of the homogenates was located in the cytosol with an optimal pH of 8.0. The activity was dependent on NAD+, and NADP+ could not substitute for NAD+. 1 mol of 20-carboxyleukotriene B4 was formed with the reduction of 2 mol of NAD+. The reaction was inhibited by pyrazole and 4-methylpyrazole, inhibitors of alcohol dehydrogenase, and by various alcohols, such as ethanol, 12-hydroxylaurate, and 20-hydroxyprostaglandin E1. Disulfiram, an inhibitor of aldehyde dehydrogenase, also inhibited the activity. These results suggest that two discrete steps catalyzed by different enzymes, alcohol dehydrogenase and aldehyde dehydrogenase, are involved in the oxidation of 20-hydroxyleukotriene B4 in rat liver cytosol. The enzyme system seems to be different from that of human neutrophils.

Animals↗
PubMed1988

Cytoplasmic pH change induced by leukotriene B4 in human neutrophils.

Leukotriene B4 induced a biphasic change in the cytoplasmic pH of human neutrophils: an initial rapid acidification followed by an alkalinization. The acidification was slightly reduced by the removal of extracellular Ca2+, but the subsequent alkalinization was not. The leukotriene B4-induced alkalinization was dependent on extracellular Na+ and pH, and was inhibited by amiloride and its more potent analogue, 5-(N,N-hexamethylene)amiloride. These characteristics indicate that the cytoplasmic alkalinization is mediated by the Na+-H+ exchange. Oxidation products of leukotriene B4, 20-hydroxyleukotriene B4, 20-carboxyleukotriene B4, and (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) also stimulated the Na+-H+ exchange, but higher concentrations were required. Treatment of the cells with pertussis toxin inhibited both phases of the leukotriene B4-induced pHi change, while cholera toxin did not affect the pHi change. The alkalinization induced by leukotriene B4 was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, but was not inhibited by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide which has a less inhibitory effect on protein kinase C. Acidification was not affected by the drugs. These findings suggest that a GTP-binding protein sensitive to pertussis toxin and protein kinase C are involved in the activation of the Na+-H+ exchange stimulated by leukotriene B4.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Intracellular pH changes of cultured bovine aortic endothelial cells in response to ATP addition.

The changes of the intracellular pH (pHi) of cultured bovine aortic endothelial cells were fluorometrically monitored using 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). A biphasic pHi change was observed by addition of ATP: an initial acidification followed by an alkalinization of about 0.2 pH unit above the resting level of pHi 7.23. The alkalinization was dependent on [Na+]o and [H+]o, and was inhibited by 5-(N,N-hexamethylene)amiloride, indicating that the alkalinization is mediated by the Na+/H+ exchanger. The 50% effective concentration of ATP was about 1.4 microM. ADP similarly induced pHi changes, whereas AMP and adenosine were inactive. The pHi changes induced by ATP were dependent on the extracellular Ca2+, and the addition of calcium ionophore A23187 induced similar pHi changes. The results indicate that ATP activates the Na+/H+ exchanger in cultured bovine aortic endothelial cells and the activation is mediated by the P2-purinergic receptor and is dependent on the extracellular Ca2+.

Adenosine Triphosphate↗
PubMed1988

Characterization of human neutrophil leukotriene B4 omega-hydroxylase as a system involving a unique cytochrome P-450 and NADPH-cytochrome P-450 reductase.

Leukotriene B4 (LTB4), a potent chemotactic agent, was catabolized to 20-hydroxyleukotriene B4 (20-OH-LTB4) by the 150,000 x g pellet (microsomal fraction) of human neutrophil sonicate. The reaction required molecular oxygen and NADPH, and was significantly inhibited by carbon monoxide, suggesting that a cytochrome P-450 is involved. The neutrophil microsomal fraction showed a carbon monoxide difference spectrum with a peak at 450 nm in the presence of NADPH or dithionite, indicating the presence of a cytochrome P-450. The addition of LTB4 to the microsomal fraction gave a type-I spectral change with a peak at around 390 nm and a trough at 422 nm, indicating a direct interaction of LTB4 with the cytochrome P-450. The dissociation constant of LTB4, determined from the difference spectra, is 0.40 microM, in agreement with the kinetically determined apparent Km value for LTB4 (0.30 microM). Such a spectral change was not observed with prostaglandins A1, E1 and F2 alpha or lauric acid, none of which inhibited the LTB4 omega-hydroxylation. The inhibition of the LTB4 omega-hydroxylation by carbon monoxide was effectively reversed by irradiation with monochromatic light of 450 nm wavelength. The photochemical action spectrum of the light reversal of the inhibition corresponded remarkably well with the carbon monoxide difference spectrum. These observations provide direct evidence that the oxygen-activating component of the LTB4 omega-hydroxylase system is a cytochrome P-450. Ferricytochrome c inhibited the hydroxylation of LTB4 and the inhibition was fortified by cytochrome oxidase. An antibody raised against rat liver NADPH-cytochrome-P-450 reductase inhibited both LTB4 omega-hydroxylase activity and the NADPH-cytochrome-c reductase activity of human neutrophil microsomal fraction. These observations indicate that NADPH-cytochrome-P-450 reductase acts as an electron carrier in LTB4 omega-hydroxylase. On the other hand, an antibody raised against rat liver microsomal cytochrome b5 inhibited the NADH-cytochrome-c reductase activity but not the LTB4 omega-hydroxylase activity of human neutrophil microsomal fraction, suggesting that cytochrome b5 does not participate in the LTB4-hydroxylating system. These characteristics indicate that the isoenzyme of cytochrome P-450 in human neutrophils, LTB4 omega-hydroxylase, is different from the ones reported to be involved in omega-hydroxylation reactions of prostaglandins and fatty acids.

Carbon Monoxide↗
PubMed1988

Characterization of the H Translocating Adenosine Triphosphatase and Pyrophosphatase of Vacuolar Membranes Isolated by Means of a Perfusion Technique from Chara corallina.

Sealed tonoplast vesicles were isolated from single cells of Chara corallina with the aid of an intracellular perfusion technique in combination with a 3/10% Percoll two step gradient centrifugation. The isolated tonoplast fraction was free from plasmalemma and chloroplasts, and showed no activities of cytochrome c oxidase, and latent IDPase, but had about 10% of the NADH-cytochrome c reductase activity. The vesicles had both ATPase and PPase activities, which could be stimulated in the presence of 10 micromolar gramicidin by 170 and 130%, respectively, demonstrating the existence of sealed vesicles. Furthermore, ATP- and PPi-dependent H(+) pumping through the membrane into the vesicles was shown. Both ATPase and PPase had pH optima around pH 8.5. At the physiological pH, 7.3, they still had more than 80% of their maximal activities. Ammonium molybdate, azide, and vanadate had no or little effect on the activities of both enzymes or their associated H(+) pumping activities. N,N'-dicyclohexylcarbodiimide inhibited the ATPase strongly (I(50) = 20 micromolar) but the PPase only weakly. The ATPase was also more sensitive to N-ethylmaleimide than the PPase. 4,4'-Stilbenedisulfonic acid affected both enzyme activities and their associated H(+) pumping activities. This is in contrast to the H(+)-PPase of higher plants which is 4,4'-stilbenedisulfonic acid insensitive.

Journal Article↗
PubMed1987

Characterization of the NADPH-dependent superoxide production activated by sodium dodecyl sulfate in a cell-free system of pig neutrophils.

The NADPH-dependent superoxide production induced by sodium dodecyl sulfate (SDS) in the sonicates of unstimulated pig neutrophils required both membrane fraction and two components of cytosol fraction. The potency of the cytosol fraction in the activation of the superoxide production could be reconstituted dose dependently by mixing two protein components with relative molecular masses of 300 kDa and 50 kDa. Another low-molecular-mass component (1.3 kDa) could substitute the 50-kDa component. In the cell-free system consisting of the 300- and 50-kDa components and the membrane fraction, the superoxide production was markedly enhanced by FAD with a required concentration for half-maximal effect of 0.16 microM and inhibited by divalent cations such as Ca2+, Ba2+, Co2+, Zn2+ and Mn2+ and not Mg2+. ATP was not necessary for the activation, indicating that protein kinases such as protein kinase C are not involved in the SDS-dependent activation of NADPH oxidase. The NADPH oxidase activated by SDS in the cell-free system was recovered in the membrane fraction, and the superoxide formation by the SDS-activated membrane exhibited a Km value for NADPH of 46 microM and optimum pH at 7.0. The formation did not require the addition of SDS and FAD to the reaction mixture and was scarcely inhibited by the divalent cations.

Animals↗
PubMed1987

Inhibition of leukotriene formation in human leukocytes by halothane.

The effects of an inhalation anesthetic, halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the formation of 5-lipoxygenase metabolites such as leukotriene B4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-isomers of leukotriene B4 and leukotriene C4 were studied in human leukocytes stimulated with calcium ionophore A23187. Halothane inhibited the formation of all these metabolites dose dependently and the formation was restored by removal of the drug. The anesthetic also reversibly inhibited the release of [3H]arachidonic acid from neutrophils with a half-inhibition concentration of less than 0.19 mM. The formation of 5-lipoxygenase metabolites was not inhibited by the anesthetic when leukocytes were stimulated with the ionophore in the presence of exogenous arachidonic acid. These observations indicate that the inhibitory effect of halothane on the formation of 5-lipoxygenase metabolites in leukocytes is mainly due to the inhibition of arachidonic acid release.

Arachidonate 5-Lipoxygenase↗
PubMed1987

NADPH-binding component of the superoxide-generating oxidase in unstimulated neutrophils and the neutrophils from the patients with chronic granulomatous disease.

The NADPH-binding component of the neutrophil superoxide-generating oxidase was studied in the particulate oxidase fractions obtained from the neutrophils of normal and chronic-granulomatous-disease (CGD) patients. The molecular mass of the NADPH-binding component of the stimulated human neutrophils, which was labelled with the 2',3'-dialdehyde derivative of NADPH and sodium cyanoboro[3H]hydride, was 66 kDa. The 66 kDa component was also labelled in monocytes, but not in red blood cells, platelets and lymphocytes. The particulate oxidase fractions obtained from the patients with CGD had a diminished amount of FAD, whether they contained cytochrome b558 or not. The fractions labelled with the NADPH analogue showed that CGD patients had the NADPH-binding component in the neutrophils. The molecular mass of the component was identical with that of the normal neutrophils. The patients are thought to have an intact NADPH-binding domain of the oxidase in the neutrophils in spite of a diminished amount of FAD in the particulate fractions. The component of the oxidase in the resting neutrophils was also labelled with the analogue. The molecular mass of the component in the resting neutrophils was identical with that of the stimulated neutrophils, and the component was not phosphorylated during the activation process. These results indicate that the NADPH-binding component of the oxidase, which is specific to phagocytes, is present in the resting neutrophils and that the component does not change with respect to molecular mass during the activation process.

Cytochrome b Group↗