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Biomedical subjects

S Minakami

Publications and source records attributed to S Minakami.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1987

Neutrophils become refractory to phorbol myristate acetate when treated with Ca2+-ionophore and Ca2+.

Human neutrophils deprived of divalent cations by treatment with ionophore A23187 in the presence of ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) showed superoxide release when they were preincubated with calcium and then treated with the ionophore. The release was not observed when the ionophore was added first and then calcium was added more than 5 min later. The absence of the release in this case can be ascribed to a refractoriness of the cells to stimuli, because the cells did not release superoxide on stimulation with phorbol myristate acetate (PMA). The cells pretreated with either calcium or the ionophore alone did release superoxide on addition of PMA. The refractoriness of the cells to PMA depended on the concentrations of calcium and the ionophore and on the time interval between the two treatments. Calcium could be replaced with Cd2+ but not with Mg2+, Ba2+, or Sr2+. The release of granular enzymes was observed when the depleted cells were pretreated with the ionophore and then with calcium. These observations indicate that calcium has dual effects on the superoxide release of neutrophils, i.e., it stimulates the cells and makes them refractory to stimuli, depending on the time interval after the addition of the ionophore, and it also regulates the enzyme release by a different mechanism.

Aminoquinolines↗
PubMed1987

Monoclonal antibody 7D5 raised to cytochrome b558 of human neutrophils: immunocytochemical detection of the antigen in peripheral phagocytes of normal subjects, patients with chronic granulomatous disease, and their carrier mothers.

We have established a monoclonal hybridoma clone that produces IgG1 against the cytochrome b558 of human neutrophils. The antibody 7D5, secreted by the hybridoma, bound to solubilized cytochrome b of the neutrophils but not to other proteins such as hemoglobin, myeloperoxidase, and pig cytochrome P-450. Immunocytochemical studies of normal human peripheral blood showed that 7D5 bound to neutrophils and monocytes but not to lymphocytes or erythrocytes. The neutrophils of male patients but not of a female patient with chronic granulomatous disease lacked the antigen of 7D5 as well as the absorption spectrum for cytochrome b558. A mosaic of the antigen-positive and -negative neutrophils was observed in mothers of the male patients. These biochemical and immunocytochemical results indicate that 7D5 is a specific antibody against cytochrome b558 of human phagocytes.

Antibodies, Monoclonal↗
PubMed1986

Inhibition of neutrophil superoxide formation by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and inhibitor of protein kinase-C.

Superoxide formation of human neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine, or calcium ionophore A23187 was inhibited by pretreatment of the cells with 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 the protein kinase-C. H-7 also inhibited superoxide formation of PMA-activated cytoplasts, which lack nuclei and granules. The phosphorylation of proteins induced by PMA in the cytoplasts as well as the intact neutrophils was also inhibited by preincubation with H-7. Among several phosphoproteins affected by H-7, one protein with a molecular weight of 19,000 (pI = 4.9) was inhibited markedly. N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide did not inhibit the phosphorylation of proteins induced by PMA. These findings support the possibility that the protein kinase-C is involved in the activation process of superoxide formation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1986

Calcium channel antagonist induced inhibition of superoxide production in human neutrophils. Mechanisms independent of antagonizing calcium influx.

Three calcium channel antagonists, verapamil, diltiazem and nisoldipine, inhibited superoxide production in human neutrophils that were stimulated by phorbol 12-myristate 13-acetate (PMA) in a buffered saline lacking calcium. Concentrations of these drugs giving 50% control activity (IC50) were 0.3, 0.45 and 0.01 mM respectively. This inhibition was also observed in the presence of ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) and was not reversed by the addition of calcium. This suggests that calcium channel antagonists inhibited superoxide production independently of extracellular calcium. These calcium channel antagonists inhibited the mobilization of membrane-associated calcium, and protein phosphorylation probably catalyzed by C-kinase, both of which are thought to be involved in the signal transmission for the induction of superoxide production. Calcium channel antagonists also inhibited NADPH oxidase, responsible for superoxide production, with IC50 = 0.5, 3 and more than 0.08 mM, respectively, for verapamil, diltiazem and nisoldipine. The results indicate that calcium channel antagonists inhibit superoxide production by affecting not only the catalytic activity by also the activation of NADPH oxidase. Inhibition of superoxide production by calcium channel antagonists suggests that these antagonists do not affect cell functions merely by affecting calcium influx.

Calcium↗
PubMed1986

NADPH oxidase of guinea-pig macrophages catalyses the reduction of ubiquinone-1 under anaerobic conditions.

The stimulation-specific NADPH-dependent reduction of ubiquinone-1 (Q-1) in guinea-pig macrophages was studied. The activity was due neither to any modified product of the phagocytosis-specific NADPH oxidase nor to non-specific diaphorases of the cells, since the activity was measured in sonicated or detergent-disrupted cells by subtracting the activity in the resting cells from that in cells activated by phorbol 12-myristate 13-acetate. The activity was not mediated by superoxide anions, since strict anaerobic conditions were employed. The anaerobic reduction of Q-1 was NADPH-specific, like superoxide formation under aerobic conditions, and its maximal velocity was also essentially the same as that of superoxide formation. The oxidase does not directly reduce Q-1 under aerobic conditions [Nakamura, Murakami, Umei & Minakami (1985) FEBS Lett. 186, 215-218], and the electron transfer from NADPH to cytochrome c by the oxidase under aerobic conditions was not enhanced by the addition of Q-1. The observations indicate that the phagocytosis-specific NADPH oxidase reduces Q-1 and that oxygen competes with the reduction of Q-1. Q-1 seems to accept electrons not from the intermediary electron carriers of the oxidase but from the terminal oxygen-reducing site of the enzyme.

Anaerobiosis↗
PubMed1986

NADPH binding component of neutrophil superoxide-generating oxidase.

The 2',3'-dialdehyde derivative of NADPH was used as an affinity labeling reagent of a solubilized NADPH-dependent superoxide-generating oxidase preparation of pig neutrophils. The analogue served as both an electron donor and a competitive inhibitor of the NADPH oxidase against NADPH. The apparent Michaelis constant (Km) for the derivative (31 microM) was essentially the same as that for NADPH (33 microM). The activity of the superoxide formation in the presence of 2',3'-dialdehyde NADPH was about a half of that in the presence of NADPH. Incubation of the enzyme with the derivative inactivated the superoxide-generating activity and the inactivation was prevented by the addition of NADPH. We performed the labeling of the oxidase preparation with 2',3'-dialdehyde NADPH and sodium cyanoboro[3H]hydride, based on the above results. A protein of 66,000 daltons was selectively labeled among more than 20 bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were visualized with Coomassie Brilliant Blue. The protein was not labeled when the oxidase preparation was pretreated with p-chloromercuribenzoate or it was labeled in the presence of excess NADPH. The protein is suggested to be the NADPH binding component of the neutrophil superoxide-generating oxidase system.

Affinity Labels↗
PubMed1986

A simultaneous quantitation of leukotriene B4 and its omega-oxidized products by gas chromatography-mass spectrometry.

We developed a highly sensitive and specific method for the simultaneous quantitation of leukotriene B4 (LTB4) and its omega-oxidized metabolites (20-hydroxy-LTB4 and 20-carboxy-LTB4) by mass fragmentography using deuterated compounds as internal standards. The ions produced by the cleavage of the C12-13 bond of the methyl ester dimethylisopropylsilyl ether derivatives of LTB4 and its metabolites were measured by selective ion monitorings. The detection limit of LTB4 was less than 10 pg and about 100-fold lower than that by high performance liquid chromatography. By using this method, the synthesis and further metabolism of LTB4 in human polymorphonuclear leukocytes were investigated.

Gas Chromatography-Mass Spectrometry↗
PubMed1986

Cinchocaine and amethocaine inhibit activation and activity of superoxide production in human neutrophils.

Local anaesthetics, cinchocaine and amethocaine, inhibited superoxide production in human neutrophils stimulated by phorbol ester, with IC50 (concentration of a drug giving 50% activity of control) values of 0.2 and 0.6 mmol litre-1, respectively. These anaesthetics inhibited protein (de)phosphorylation and mobilization of membrane-associated calcium, both of which are probable signal transmission mechanisms for the induction of superoxide production. Local anaesthetics also inhibited NADPH oxidase, responsible for superoxide production, with IC50 = 0.3 and 0.9 mmol litre-1, respectively. The results indicate that cinchocaine and amethocaine inhibit superoxide production in human neutrophils by affecting not only the catalytic activity, but also the activation, of NADPH oxidase.

Blood Proteins↗
PubMed1986

2,6-Dichlorophenolindophenol-reducing activity of phagocytosis-associated NADPH oxidase.

The 2,6-dichlorophenolindophenol (DCIP)-reducing activity of the phagocytosis-associated NADPH oxidase was investigated using homogenates and a membrane fraction (F2) of elicited guinea pig peritoneal macrophages stimulated by phorbol myristate acetate. Essentially all of the stimulation-specific DCIP reduction under aerobic conditions could be inhibited when high concentrations of superoxide dismutase (SOD), about 10 times those usually used to inhibit the superoxide (O-2)-mediated cytochrome c reduction, were used. SOD inhibited the DCIP reduction by chemically generated O2- in the same manner as the stimulation-specific DCIP reduction by the macrophage F2, and the concentration of SOD necessary for 50% inhibition was about 10 times that for the reduction of cytochrome c. Under anaerobic conditions, however, the NADPH oxidase could reduce DCIP, though the rate was slow because we could not use a sufficiently high DCIP concentration. The observations indicate that the NADPH oxidase preferentially reduces oxygen under aerobic conditions, though the oxidase can reduce DCIP in the anaerobic state.

2,6-Dichloroindophenol↗
PubMed1986

Spontaneous induction of superoxide release and degranulation of neutrophils in isotonic potassium medium: the role of intracellular calcium.

When guinea pig peritoneal neutrophils were suspended in the isotonic medium of potassium, rubidium, and cesium ions at 37 degrees C, the cells released superoxide, while low activity was observed in the isotonic medium of sodium and lithium ions. The activity induced in the potassium medium was enhanced by potassium-ionophores, valinomycin, and gramicidin, and decreased by a potassium channel blocker, 4-aminopyridine. The superoxide-releasing activity was not affected by the presence or absence of extracellular calcium but was inhibited by an intracellular calcium antagonist-8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate(TMB-8) with the half-inhibition concentration of 50 microM. The release of granular enzymes, lysozyme and beta-glucuronidase, was also induced in the isotonic potassium medium in the absence of extracellular calcium and inhibited by TMB-8. A remarkable elevation of the intracellular free calcium concentration in neutrophils, which was monitored by quin-2 fluorescence, was found when the cells were added to the potassium medium without calcium. The elevation was inhibited by the addition of TMB-8. These observations suggest that calcium mobilization from intracellular storage sites, not an influx of calcium from the extracellular medium, causes the release of superoxide and the granular enzymes in isotonic potassium medium.

Animals↗
PubMed1986

NADPH-dependent superoxide-forming oxidase in phagocytic vesicles of human monocytes.

Phagocytic vesicles with superoxide-forming NADPH oxidase activity were obtained from human monocytes phagocytosing oil droplets. The superoxide-forming activity in the monocyte vesicles increased for the first 5 min during incubation with oil droplets and remained constant for 30 min. NADPH-dependent activities of 2,6-dichlorophenol-indophenol (DCIP) reduction and ubiquinone-1 (Q1) reduction were found in the vesicles and the activities were closely associated with the superoxide-forming oxidase. The values of apparent Km for NADPH of these three activities were essentially the same and the activities were inhibited with a similar pattern by p-chloromercuribenzoate and a cationic detergent, cetyltrimethylammonium bromide. The activities were extremely labile and the DCIP reductase activity was most labile. The superoxide-forming oxidase and the Q1 reductase could be extracted with a mixture of deoxycholate and Tween-20. The extracted activities were not enhanced by the addition of FAD.

2,6-Dichloroindophenol↗
PubMed1986

Inhibition of superoxide production and Ca2+ mobilization in human neutrophils by halothane, enflurane, and isoflurane.

The inhibitory effects of three inhalation anesthetics, i.e., halothane, enflurane, and isoflurane, on superoxide production and the intracellular mobilization of calcium in human neutrophils were studied. The superoxide production induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) was inhibited by the anesthetics, but the binding of FML[3H]P to the cells and the superoxide-forming NADPH oxidase of the phagocytic vesicles were not inhibited. The inhibition of the cellular superoxide production was partially reversed by the addition of a calcium ionophore, A23187. The increase in intracellular free calcium monitored by a calcium-sensitive fluorescent probe, quin-2 and the release of calcium from hydrophobic environment monitored by chlortetracycline were inhibited dose dependently by the anesthetics. These observations suggest that decreased mobilization of intracellular Ca2+ is one of the mechanisms by which the anesthetics inhibited the superoxide production of human neutrophils stimulated by FMLP.

Calcimycin↗
PubMed1985

NAD+-dependent conversion of 20-OH-LTB4 to 20-COOH-LTB4 by a cell-free system of human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes, but not mononuclear leukocytes, platelets, or erythrocytes, oxidized 20-hydroxy-leukotriene B4 (20-OH-LTB4) to 20-carboxy-LTB4 (20-COOH-LTB4). 20-OH-LTB4 was quantitatively converted to 20-COOH-LTB4 by the sonicate of polymorphonuclear leukocytes in the presence of NAD+, with an optimal pH of about 7.9. NADP+ could not replace NAD+. The conversion was not inhibited by 2 mM pyrazole, a potent inhibitor of alcohol dehydrogenase. When LTB4 was incubated with the sonicate in the presence of NADPH and NAD+, 20-OH-LTB4 and 20-COOH-LTB4 appeared concomitantly with the disappearance of LTB4.

Cell-Free System↗
PubMed1985

Pertussis toxin inhibits intracellular pH changes in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine.

Changes of intracellular pH in human neutrophils were monitored by 9-aminoacridine fluorescence. Both initial acidification and subsequent alkalinization phases induced by a chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine were dependent on the extracellular Ca2+-concentrations, and a calcium ionophore, A-23187 similarly induced the pH-changes. Pertussis toxin inhibited the pH-changes induced by the peptide while cholera toxin did not. The pH-changes induced by A-23187 were not affected by the toxins. The results suggest that the inhibitory guanine-nucleotide regulatory protein and Ca2+ are involved in the pH-changes induced by the peptide.

Adult↗