Biomedical subjects

S Minakami

Publications and source records attributed to S Minakami.

At least 37 records · Page 2Linked to original sources

PubMed1992

Role of Mg2+ in activation of NADPH oxidase of human neutrophils: evidence that Mg2+ acts through G-protein.

The membrane fraction and three cytosolic proteins of neutrophils, p47-phox, p67-phox and a G-protein, are involved in the cell-free activation of the O2(-)-generating NADPH oxidase in the presence of SDS, though it has been controversial whether the G-protein is required or just enhancing the activity. We have used the three cytosolic factors, the solubilized membrane fraction, GTP gamma S and SDS, and found that both G-protein and GTP gamma S are essential for the activation of the NADPH oxidase. The effect of GTP gamma S is modified by Mg2+: the cations enhance the O2- generation at low concentrations of GTP gamma S, whereas they attenuate the activity at higher concentrations of GTP gamma S. In presence of 10 microM GTP gamma S, the maximal activity is observed at 0.1 microM Mg2+, which is several-fold higher than that at 1 mM Mg2+. The omission of Mg2+ followed by the chelation with EDTA results in loss of the activation, which is completely restored by the addition of Mg2+. Thus, Mg2+ seems to modulate the activation of the NADPH oxidase at the level of the G-protein.

Antibodies, Monoclonal↗

PubMed1992

Succinate-dependent lipid peroxidation and its prevention by reduced ubiquinone in beef heart submitochondrial particles.

When succinate and ADP-Fe3+ chelate were added to beef heart submitochondrial particles pretreated with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase of the mitochondrial respiratory chain, the formation of malondialdehyde was observed. No formation was observed without the pretreatment. Oxaloacetate competitively inhibited the malondialdehyde formation with an apparent Ki of 3.4 microM. The malondialdehyde formation seemed to be initiated at the location between the p-hydroxymercuribenzoate-sensitive site and the 2-thenoyltrifluoroacetone-sensitive site of the succinate dehydrogenase because it was inhibited by the mercurial. Ubiquinone-10 was rapidly destroyed during the malondialdehyde-forming reaction when it was in the oxidized form, while the ubiquinone was not destroyed and the malondialdehyde formation was abolished when about 50% of the ubiquinone in the particles was in the reduced state. These observations suggest that the succinate-dependent peroxidation is strongly controlled by the redox state of ubiquinone.

Animals↗

PubMed1992

Activation of cultured bovine aortic endothelial cells by extracellular pyrimidine triphosphate.

1. The addition of ATP to cultured bovine aortic endothelial cells induced the increase in intracellular free calcium concentration ([Ca2+]i) and thereby activated the sodium/proton exchanger and the prostacyclin production in a similar dose-dependent manner, as observed by the addition of ATP. 2. Other nucleoside triphosphates also activated the cells and the potency orders of the nucleotides were ATP greater than UTP greater than ITP greater than CTP greater than GTP for all the responses. 3. Pretreatment of the cells with UTP desensitized the response to ATP and the pretreatment of ATP desensitized the response to UTP. 4. The responses to ATP and UTP were inhibited by neither pertussis nor cholera toxin. 5. The receptor for UTP, however, may be a distinct pyrimidinoceptor different from the purinoceptor of the cells for ATP, because the 50% effective concentration of UDP was much larger than that of UTP, while ATP and ADP were essentially equipotent ligands to the endothelial cells.

Adenosine Triphosphate↗

PubMed1991

Effects of iodothyronines on chemotactic peptide-receptor binding and superoxide production of human neutrophils.

We studied the action of iodinated thyronines on the superoxide (O2-) production of human neutrophils stimulated with a chemotactic peptide N-formylmethionylleucylphenylalanine (FMLP) in vitro. L-Thyroxine and L-triiodothyronine elicited dose dependently a potent inhibitory action on the FMLP-induced O2- production with IC50 values of about 10(-6) M and 7.10(-6) M, respectively, but L-diiodothyronine did not. No difference in the inhibition was observed between the L-form and the D-form of the compounds. Inhibition of the O2- production by L-thyroxine was restored by the washing of the cells. L-Thyroxine did not affect the O2- production stimulated with either the fifth component of the complement (C5a) or phorbol 12-myristate 13-acetate. L-Thyroxine and L-triiodothyronine were found to block [3H]FMLP binding to its own receptor with IC50 values similar to those for the inhibition of the O2- production by changing the affinity for the peptide but not the number of the receptors. These results suggest that thyroxine and triiodothyronine interfere with the binding of FMLP to the receptors, leading to the inhibition of neutrophil functions, such as O2- production, and that the inhibitory effects result from extranuclear actions rather than nuclear receptor-mediated ones.

Chemoreceptor Cells↗

PubMed1991

Evidence that the decarboxylation reaction occurs before the first methylation in ubiquinone biosynthesis in rat liver mitochondria.

Biosynthesis of ubiquinone-9 was studied by incubating rat liver mitochondria with p-hydroxy[U-14C]benzoate, solanesyl diphosphate and S-adenosyl-L-methionine. When methylation reactions were inhibited by replacing S-adenosyl-L-methionine with S-adenosyl-L-homocysteine, nonaprenyl p-hydroxybenzoate and three other labeled peaks, designated as P1, P2 and P3 according to their retention times on HPLC, were observed. No carboxyl group was present in P1, P2 or P3 because the radioactivities disappeared when p-hydroxy[U-14C]benzoate was replaced by p-hydroxy[carboxyl-14C]benzoate. Compound P2 seemed to be hydroxylated but not methylated because its radioactivity markedly diminished under anaerobic conditions and the radioactivity was not incorporated into the compound from S-adenosyl-L-[methyl-3H]methionine, suggesting that P2 is 6-hydroxynonaprenylphenol. The complete correspondence of the retention times of P2 and chemically synthesized 6-hydroxynonaprenylphenol on HPLC further confirmed this possibility. P2 was a precursor of ubiquinone-9 because the radioactivity of the compound was incorporated into ubiquinone when incubated with mitochondria. The results suggest that the decarboxylation may occur prior to the first methylation in the ubiquinone biosynthesis in rat liver mitochondria, though it has been generally considered that in eukaryotes the first methylation precedes the decarboxylation.

Anaerobiosis↗

PubMed1991

Bilirubin inhibits the activation of superoxide-producing NADPH oxidase in a neutrophil cell-free system.

We studied the effect of bilirubin on the NADPH-dependent superoxide production induced by sodium dodecyl sulfate in a cell-free system consisting of the membrane and cytosolic fractions of pig neutrophils. Preincubation of the cytosolic fraction with bilirubin before the addition of sodium dodecyl sulfate resulted in the time- and dose-dependent inhibition of the superoxide production while the preincubation of the membrane fraction with the tetrapyrrole did not result in the inhibition. When the pigment was added after the initiation of the reaction, the ongoing production was not affected by the addition. Other tetrapyrroles, such as hemin, protoporphyrin and biliverdin, also inhibited the production. The results indicate that bilirubin inhibits the activation process of the superoxide producing NADPH oxidase by decreasing the potency of the cytosolic fraction and its inhibitory effect seems to be due to the hydrophobic nature of the tetrapyrrole.

Animals↗

PubMed1990

Endothelin-1 enhances superoxide generation of human neutrophils stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine.

Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing the superoxide (O2-) generation of human neutrophils, but it enhanced the O2- generation stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) about 2-fold when the cells had been preincubated with ET-1 for 10 min at 37 degrees C. The concentration at which ET-1 was 50% effective was 1 x 10(-10) M, and the maximal effect was obtained at 1 x 10(-8) M. The enhancement was observed over the range of the effective concentrations of FMLP (10(-8)-10(-6) M). ET-1 did not promote the mobilization of intracellular calcium ions and the enhancing effect of ET-1 did not change when calcium ions were depleted. These findings indicate that ET-1 is a potent modulator of human neutrophils and may thus contribute to the inflammatory process.

Calcium↗

PubMed1990

1-Methyl-4-phenylpyridinium (MPP+) induces NADH-dependent superoxide formation and enhances NADH-dependent lipid peroxidation in bovine heart submitochondrial particles.

We studied the effects of 1-methyl-4-phenylpyridinium (MPP+), a metabolite of a parkinsonism-inducing drug, on the superoxide formation and the lipid peroxidation in bovine heart submitochondrial particles. The NADH-supported formation of superoxide radicals was induced by MPP+ at the concentration which is considered to exist in mitochondria of dopamine neurons. The formation increased as the NADH-ubiquinone reductase activity was inhibited by MPP+. The NADH-supported lipid peroxidation by the particles in the presence of ADP-Fe3+ chelate was also enhanced by MPP+ at similar concentrations. The formation was inhibited by succinate and the reduction of endogenous ubiquinone seems to be related to the inhibition. A possibility was discussed that the formation of superoxide anions and the lipid peroxidation may contribute in the cytotoxicity of the drug.

1-Methyl-4-phenylpyridinium↗

PubMed1990

Oxidation of 20-hydroxyleukotriene B4 to 20-carboxyleukotriene B4 by human neutrophil microsomes. Role of aldehyde dehydrogenase and leukotriene B4 omega-hydroxylase (cytochrome P-450LTB omega) in leukotriene B4 omega-oxidation.

Leukotriene B4 (LTB4), a potent chemoattractant for leukocytes, is catabolized by human neutrophils via omega-oxidation. Neutrophil microsomes are known to oxidize 20-hydroxy-LTB4 (20-OH-LTB4) to its 20-oxo and 20-carboxy derivatives in the presence of NADPH. This activity has been ascribed to LTB4 omega-hydroxylase (cytochrome P-450LTB omega), a conclusion supported by our finding of the reversal of carbon monoxide inhibition by 450 nm light and by competitive inhibition studies. The oxidation of 20-oxo-LTB4 to 20-carboxy-LTB4 is also catalyzed by microsomes fortified with 1 mM NAD+, and this activity is not affected by cytochrome P-450LTB omega inhibitors. The evidence is compatible with involvement of a disulfiram-insensitive aldehyde dehydrogenase in this second oxidation pathway. Interaction of the two pathways is evidenced by facilitation of NADPH-dependent oxidation of 20-OH-LTB4 by the addition of NAD+. This synergism may be explained by removal of the aldehyde intermediate by the NAD(+)-dependent aldehyde dehydrogenase. Taken together with the finding that the NAD(+)-dependent activity is severalfold higher than the NADPH-dependent one, the dehydrogenase may be important in the oxidation of 20-OH-LTB4 to 20-carboxy-LTB4.

Aldehyde Dehydrogenase↗

PubMed1990

Formation of 20-oxoleukotriene B4 by an alcohol dehydrogenase isolated from human neutrophils.

When 20-hydroxyleukotriene B4 (20-OH-LTB4) is incubated at pH 10.5 in the presence of NAD+ with an alcohol dehydrogenase isolated from human neutrophils, a polar product is formed as detected on reverse-phase high-performance liquid chromatography (RP-HPLC). The product is identified as 20-oxo-LTB4 (20-CHO-LTB4) on the basis of its co-elution with the authentic compound on HPLC, ultraviolet spectrometry and gas chromatography-mass spectrometry. The 20-CHO-LTB4-forming activity requires NAD+, but NADP+ scarcely replaces NAD+. The apparent Km for 20-OH-LTB4 is 83 microM and the Vmax is 2.04 mumol/min per mg of protein. The activity is inhibited by omega-hydroxy fatty acids such as 12-hydroxylauric acid, 16-hydroxypalmitic acid and 12(S), 20-dihydroxyeicosatetraenoic acid, but not by 4-methylpyrazole. At pH 7.0 with NADH, the purified dehydrogenase catalyzes the reverse reaction, the reduction of 20-CHO-LTB4 to 20-OH-LTB4.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1990

Reconstitution of the partially purified membrane component of the superoxide-generating NADPH oxidase of pig neutrophils with phospholipid.

The NADPH-dependent superoxide-generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell-free system. The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat-germ-agglutinin-agarose column chromatography. The chromatography resulted in loss of the O2--generating activity in the cell-free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N-acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre-mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent-free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 +/- 0.53 nmol/mg protein (mean +/- SD, n = 5). The potency of fraction B in the reconstitution of the O2--generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2--generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2--generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p-chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0-7.5. The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 +/- 3.5 mumol O2-.min-1.mg-1 membrane-derived protein (mean +/- SD, n = 5) which shows that the membrane component was purified about 100-fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2--generating NADPH oxidase and its activation in the cell-free system requires the reconstitution with phospholipids.

Animals↗

PubMed1990

Fragmentation of re-formation of mitotic Golgi apparatus detected by a centrifugal method.

The fragmentation/re-formation process of the Golgi apparatus during mitosis was studied by flotation centrifugation in a stepwise sucrose density gradient. The mitotic Golgi fraction was obtained from Chinese hamster ovary cells synchronized with thymidine and nocodazole. The Golgi apparatus detected by a marker enzyme, galactosyltransferase, was separated into two peaks by the flotation centrifugation. The amount of the Golgi recovered at the lower density peak was less in the mitotic cells than in the interphase cells. The separation profile of the mitotic Golgi returned to that of the interphase Golgi by further incubation of the mitotic cells. The re-formation of the fragmented Golgi was inhibited by nocodazole and vinblastine, but not by actinomycin D and cycloheximide.

Animals↗

PubMed1990

Leukotriene B4 omega-hydroxylase in rat liver microsomes: identification as a cytochrome P-450 that catalyzes prostaglandin A1 omega-hydroxylation, and participation of cytochrome b5.

The omega-hydroxylation of leukotriene B4 (LTB4) by rat liver microsomes requires NADPH and molecular oxygen, suggesting that the hydroxylation is catalyzed by a cytochrome P-450 (P-450)-linked monooxygenase system. The reaction is inhibited by CO, and the inhibition is reversed by irradiation of light at 450 nm in a light-intensity-dependent manner. The extent of the reversal is strongly dependent on the wavelength of the light used, the 450-nm light is most efficient. The finding provides direct evidence for the identification of the LTB4 omega-hydroxylase as a P-450. The P-450 seems to be also responsible for prostaglandin A1 (PGA1) omega-hydroxylation, but not for lauric acid omega-hydroxylation. The LTB4 omega-hydroxylation is competitively inhibited by PGA1, but not affected by lauric acid. The Ki value for PGA1 of 38 microM agrees with the Km value for PGA1 omega-hydroxylation of 40 microM. LTB4 inhibits the PGA1 omega-hydroxylation by rat liver microsomes in a competitive manner with the Ki of 43 microM, which is consistent with the Km for the LTB4 omega-hydroxylation of 42 microM. An antiserum raised against rabbit pulmonary PG omega-hydroxylase (P-450p-2) inhibits slightly the omega-hydroxylations of LTB4 and PGA1, while it has stronger inhibitory effect on lauric acid omega-hydroxylation. In addition to NADPH-cytochrome P-450 reductase, cytochrome b5 appears to participate in the LTB4 omega-hydroxylating system, since the reaction is inhibited by an antibody raised against the cytochrome b5 as well as one raised against the reductase.

Animals↗

PubMed1990

An intermediate of ubiquinone biosynthesis exists in the microsomal fraction of HepG2 cells.

We analyzed lipids extracted from human hepatoma HepG2 cells using a high performance liquid chromatograph equipped with a reversed phase column and found a compound with a mass spectrum showing certain diagnostic ion fragments of 1-methoxy-5-polyprenyl-phenol, a known intermediate of ubiquinone biosynthesis. Universally radiolabeled [14C]-p-hydroxybenzoate, a precursor of ubiquinone, was incorporated into the compound on incubation with the cells, suggesting that the compound is a precursor of ubiquinone. The presence of the compound in the microsomal fraction of HepG2 cells was not due to contamination by the mitochondrial fraction because the activity of succinate-cytochrome c reductase in the microsomal fraction was below 1% of that in the mitochondrial fraction, whereas the contents of ubiquinone and the compound in the former were 4.6 and 7.8% of those in the latter, respectively. These results support the hypothesis that ubiquinone biosynthesis might occur in microsomes as well as mitochondria.

Chromatography, High Pressure Liquid↗

PubMed1990

Antibodies against rabbit omega-hydroxylases of prostaglandins and fatty acids cross-react with leukotriene B4 omega-hydroxylase in human neutrophils (cytochrome P-450LTB omega).

Leukotriene B4 (LTB4) omega-hydroxylase activity in human neutrophil microsomes was significantly inhibited by antisera against three rabbit omega-hydroxylase P-450s, lung prostaglandin omega-hydroxylase (P-450p-2), small intestine prostaglandin A omega-hydroxylase (P-450ia), and kidney fatty acid omega-hydroxylase (P-450kd). In contrast, the activity is not affected by antibodies raised against the phenobarbital-inducible forms of P-450s from both rabbits and rats. These findings suggest that the LTB4 omega-hydroxylase (P-450LTB omega) is structurally related to a group of rabbit omega-hydroxylase P-450s. The antiserum raised against P-450p-2 also inhibited the NADPH-dependent oxidation of 20-hydroxy LTB4 to 20-oxo LTB4 and 20-carboxy LTB4 by the microsomes, supporting that P-450LTB omega is able to catalyze the subsequent oxidation of 20-hydroxy LTB4 as well as the omega-hydroxylation of LTB4.

Animals↗

PubMed1989

Complementary DNA for the mouse homolog of the small subunit of human cytochrome b558.

Cytochrome b558, a heterodimeric protein, is considered to be a component of the microbicidal oxidase in phagocytes. We have isolated a cDNA clone for the mouse homolog of the small subunit of human cytochrome b558. Comparison of the predicted amino acid sequence with that of the human protein reveals a high degree of homology (87%). As expected, the mRNA is expressed abundantly in peritoneal neutrophils and, to a lesser extent, in peritoneal macrophages. The transcript is also abundant in the mouse kidney. Although the spleen and small intestine contain considerable amounts of the mRNA, only trace amounts are detected in the brain, liver, testis and heart. The distribution, especially its high expression in the kidney, may suggest another function of the small subunit.

Amino Acid Sequence↗

PubMed1989

Involvement of calcium and protein kinase C in the activation of the Na+/H+ exchanger in cultured bovine aortic endothelial cells stimulated by extracellular ATP.

We have studied the activation of the Na+/H+ exchanger which leads to the intracellular alkalinization in cultured bovine aortic endothelial cells stimulated by extracellular ATP. The alkalinization induced by ATP was largely dependent on extracellular Ca2+ and the rate of alkalinization was decreased by about 60% in the absence of extracellular Ca2+. ATP caused a rapid and transient increase and a subsequent sustained increase of the intracellular Ca2+ concentration ([Ca2+]i) in the Ca2+ buffer, while only the rapid and transient increase of [Ca2+]i was observed in the absence of extracellular Ca2+. The Ca2+-depleted cells prepared by incubation in Ca2+-free buffer containing 0.1 mM EGTA showed only a slight increase of [Ca2+]i with no alkalinization on stimulation by ATP. The alkalinization was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, but not by another isoquinoline analogue (HA 1004), which has a less inhibitory effect on the kinase. Phorbol 12-myristate 13-acetate also induced the alkalinization by the activation of the Na+/H+ exchanger. Neither dibutyryl cyclic AMP nor dibutyryl cyclic GMP affected the alkalinization induced by ATP. Treatment of the cells by pertussis and cholera toxins had no effect on the alkalinization. The results suggest that the increase in [Ca2+]i is essential for the ATP-induced activation of the Na+/H+ exchanger in cultured bovine aortic endothelial cells and a protein kinase C-dependent pathway is involved in the activation.

Adenosine Triphosphate↗

PubMed1989

Extracellular ATP triggers superoxide production in human neutrophils.

The effects of ATP on the concentration of cytosolic calcium [( Ca2+]i) were examined with respect to early events associated with activation of the superoxide (O2-)-generating system in human neutrophils. Addition of ATP to cytochalasin B-treated neutrophils resulted in two sequential increase in [Ca2+]i: an initial phase presumably related to the mobilization of Ca2+ from intracellular stores and a second phase dependent upon the presence of extracellular Ca2+. The second phase was associated with an increase in the rate of O2- production, which also required the presence of extracellular Ca2+. The results suggest that increased Ca2+ influx may act to trigger a cascade of Ca2+-sensitive events, leading to stimulated O2-production.

Adenosine Triphosphate↗