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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of the antiarrhythmic drugs lidocaine, quinidine and procainamide on macrophage function was investigated in RAW 264.7 mouse monocytic macrophage cell. Cells stimulated by either zymosan or phorbol ester were found to generate both superoxide (O2-) and H2O2. The production of O2 was detected as superoxide dismutase inhibitable ferricytochrome c reduction. H2O2 production was monitored in both chemical and flow cytometric fluorescent assays. Although all three drugs inhibited both O2 and H2O2 release in a dose-dependent manner, only quinidine was found to have significant inhibitory effects. The amounts of quinidine required to cause a 50% inhibition in O2 production in zymosan and phorbol ester stimulated cells were found to be 250 microM and 300 microM, respectively and the amounts required to cause one-half optimum levels of H2O2 production in these cells were found to be 50 microM and 100 microM, respectively. The effect of these drugs on O2 producing NADPH oxidase was investigated and only procainamide was found to have a significant effect (p < 0.001) in inhibiting the oxidase activity. Lidocaine and quinidine had no significant effect on the activation of the respiratory burst oxidase. A sensitive and convenient 'differential phagocytosis' assay was devised on the basis of number of particles engulfed by individual phagocytes using flow cytometric techniques. It appears to be remarkably free of interference and was applied to investigate the role of antiarrhythmic drugs on the phagocytosis of fluorescent latex beads. All three antiarrhythmic drugs inhibited phagocytosis of latex beads in a dose dependent manner irrespective of the number of particles phagocitized by the cells. The results of these studies do not conclusively establish a mechanism of action of these drugs on the generation of O2 and H2O2 by stimulated macrophages; nevertheless, it is interesting that all three drugs inhibited the phagocytic activity.
Mol Cell Biochem 1994 Mar 30
PMID:Impairment of raw 264.7 macrophage function by antiarrhythmic drugs. 796 98

We have observed that lucigenin-derived chemiluminescence (CL) measures superoxide anion (O2-) production from two major cellular compartments in rat alveolar macrophages (AMs): extracellular O2- produced by NADPH oxidase; and intracellular O2- produced by the reduction of molecular oxygen by the mitochondrial electron transport chain. Although the treatment of AMs with 30 ng/mL 12-O-tetradecanoyl phorbol-13-acetate (TPA) increases the production of O2- by NADPH oxidase, the net result of TPA addition monitors is a decrease in lucigenin-derived CL resulting from inhibition of mitochondrial-derived O2- production. Since protein kinase C (PKC) has been shown to be the cellular receptor through which TPA mediates its effects, it was postulated that activation of PKC inhibits mitochondrial-derived O2- production as measured by lucigenin-derived CL. Studies performed with 50 microM H-7 (a PKC inhibitor) support this hypothesis by blocking the inhibition of mitochondrial-derived CL by TPA, while HA-1004, a negative control for H-7, had no effect on the system. These results suggest that mitochondrial respiration may be modulated by the actions of PKC. Moreover, this observation suggests a novel mechanism whereby chemicals which activate PKC may affect cellular function through modulation of mitochondrial activity.
Res Commun Mol Pathol Pharmacol 1994 Aug
PMID:Inhibition of mitochondrial superoxide generation in rat alveolar macrophages by 12-O-tetradecanoylphorbol-13-acetate: potential role of protein kinase C. 799 56

Phagocytes are able to generate reactive oxygen species by an activatable NADPH oxidase system. We investigated the inhibition of NADPH oxidase activation by a methoxy-substituted catechol, apocynin. Oxygen uptake by neutrophils incubated with 300 microM apocynin was completely inhibited at 7 min after addition of serum-treated zymosan (STZ), with a lagtime of inhibition of 2 to 3 min. The lagtime of effect of apocynin in neutrophils relatively deficient of myeloperoxidase was about 50% longer when compared with normal cells. Inhibition of the STZ-induced respiratory burst by apocynin was also observed in human eosinophils but not in human alveolar macrophages. Immunoblots of neutrophil membranes, isolated at 2 and 7 min after STZ stimulation of neutrophils, demonstrated translocation of the cytosolic oxidase components p47-phox and p67-phox to the membrane fraction. Translocation at 7 min after STZ stimulation was markedly reduced when the neutrophils had been incubated with 300 microM apocynin, but translocation was normal after 2 min of stimulation. These properties suggest that apocynin is an intracellular inhibitor of the assembly of NADPH oxidase in neutrophils and eosinophils and that apocynin requires conversion by peroxidases to exert its inhibitory effect. The capacity of neutrophils for intracellular killing of Staphylococcus aureus was not affected by apocynin. The potential therapeutic value of apocynin was demonstrated in vitro by its ability to protect secretory leukocyte proteinase inhibitor from oxidative inactivation by neutrophils.
Am J Respir Cell Mol Biol 1994 Jul
PMID:Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. 801 41

Protein X from Azotobacter vinelandii has recently been shown to be either a NADPH oxidase or a NADP+ reductase that interacts specifically with ferredoxin I. Single crystals have been obtained by vapor diffusion from polyethylene glycol 4000 solutions containing 100 mM citrate buffer (pH 5.5). The crystals belong to space group P2(1)2(1)2 with unit cell constants a = 68.9 A, b = 76.9 A, c = 52.8 A and one molecule (M(r) 29,000) per asymmetric unit. The crystals diffract to 2.5 A resolution.
J Mol Biol 1994 Aug 26
PMID:Diffraction quality crystals of protein X from Azotobacter vinelandii. 805 82

Fluorescence emission intensity of 1,3-diphenylisobenzofuran incorporated in polymorphonuclear granulocytes plasma membranes was investigated in basal conditions and during stimulation with different activators. Phorbol myristate acetate, known as the most effective "oxygen burst" inducer, produced a larger decrease in 1,3-diphenylisobenzofuran fluorescence intensity than 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) and N-formyl-methionyl-leucyl-phenylalanine, known as weak stimulants of oxygen uptake. Diphenyl iodonium an inhibitor of leukocyte NADPH oxidase, and the singlet oxygen selective trap alpha-terpinene inhibited the quenching effect of phorbol myristate acetate on 1,3-diphenylisobenzofuran fluorescence. These data suggest formation of singlet oxygen in activated leukocytes and demonstrate that measurement of 1,3-diphenylisobenzofuran fluorescence intensity provides a new sensitive method of detection of neutrophils activation.
Biochem Mol Biol Int 1994 Apr
PMID:A sensitive detection of neutrophil activation by fluorescence quenching of membrane inserted singlet oxygen probe. 806 26

Activation of the NADPH oxidase of phagocytic cells requires the action of Rac2 or Rac1, members of the Ras superfamily of GTP-binding proteins. Rac proteins are active when in the GTP-bound form and can be regulated by a variety of proteins that modulate the exchange of GDP for GTP and/or GTP hydrolysis. The p190 Rac GTPase Activating Protein (GAP) inhibits human neutrophil NADPH oxidase activity in a cell-free assay system with a K1 of approximately 100 nM. Inhibition by p190 was prevented by GTP gamma S, a nonhydrolyzable analogue of GTP. Similar inhibition was seen with a second protein exhibiting Rac GAP activity, CDC42Hs GAP. The effect of p190 on superoxide (O2-) formation was reversed by the addition of a constitutively GTP-bound Rac2 mutant or Rac1-GTP gamma S but not by RhoA-GTP gamma S. Addition of p190 to an activated oxidase produced no inhibitory effect, suggesting either that p190 no longer has access to Rac in the assembled oxidase or that Rac-GTP is not required for activity once O2- generation has been initiated. These data confirm the role of Rac in NADPH oxidase regulation and support the view that it is the GTP form of Rac that is necessary for oxidase activation. Finally, they raise the possibility that NADPH oxidase may be regulated by the action of GAPs for Rac proteins.
Mol Biol Cell 1993 Nov
PMID:Regulation of NADPH oxidase activity by Rac GTPase activating protein(s). 830 40

Local production of reactive oxygen intermediates, e.g., superoxide anion, by tumor promoter-stimulated inflammatory macrophages (MPs) may contribute significantly to tumor development in classical models of two-stage chemical-induced carcinogenesis in murine skin. In the studies reported herein, peritoneal MPs elicited from phorbol-ester-sensitive SENCAR mice demonstrated a time- and dose-dependent release of superoxide anion (4-6 nmol/10(6) cells) when stimulated by 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro; MP superoxide response was significantly inhibited (50-70%) by preincubation with 40 microM 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), a protein-kinase inhibitor. Alternatively, TPA-stimulated MPs derived from relatively resistant B6C3F1 mice generated negligible superoxide under the same conditions. A similar strain-dependent induction of superoxide was observed when MPs were stimulated with thapsigargin (TG), a tumor promoter previously shown to act independently of protein kinase C (PKC). TG-stimulated SENCAR MPs released a significant amount of superoxide (2-3 nmol/10(6) cells) that was not inhibited by H-7; MPs from B6C3F1 mice demonstrated negligible stimulation by TG. Preincubation of SENCAR MPs with 100 microM dibromoacetophenone, an inhibitor of phospholipase A2, completely suppressed the superoxide induced by TPA and TG stimulation. Like TPA, 50 microM 1-oleoyl-2-acetylglycerol, a diacylglycerol analogue and PKC activator, also induced a significant amount of superoxide from SENCAR MPs only. In parallel with the superoxide findings, TPA and TG stimulated significantly greater [3H]arachidonic acid release from prelabeled SENCAR MPs (a 32% and 48% increase, respectively, over unstimulated controls) relative to MPs from B6C3F1 mice. Two-dimensional gel-electrophoretic analysis indicated that TPA-induced phosphorylation of a 47-kDa protein (a presumed substrate for PKC previously linked to NADPH oxidase activation in guinea pig and human polymorphonuclear leukocytes) occurred in MPs from both SENCAR and B6C3F1 mice. Therefore, arachidonic acid production may be a common biochemical pathway by which phorbol-ester--and non-phorbol-ester--type tumor promoters activate MPs in SENCAR mice; such a response may be "permissive" for additive (or synergistic) interactions with PKC-driven signal pathways.
Mol Carcinog 1993
PMID:Induction of superoxide by 12-O-tetradecanoylphorbol-13-acetate and thapsigargin, a non-phorbol-ester-type tumor promoter, in peritoneal macrophages elicited from SENCAR and B6C3F1 mice: a permissive role for the arachidonic acid cascade in signal transduction. 838 57

Administration of nifedipine to mice over a period of six months caused a significant (p < 0.05) decrease in neutrophilic functions viz superoxide generation, coupled to NADPH oxidase activity as well as NADPH production by HMP shunt. Properties like chemotaxis and phagocytosis showed a similar decrease. From this study, it is seen that nifedipine causes neutrophil functional abrogation which is therefore an apparent concern for the prolonged usage of the drug. However, relevance of the mouse model to clinical situation needs further investigation.
Mol Cell Biochem 1993 Mar 10
PMID:Effect of nifedipine administration on the functional capacity of neutrophils. 838 90

Rac1 and Rac2 are closely related, low molecular weight GTP-binding proteins that have both been implicated in regulation of phagocyte NADPH oxidase. This enzyme system is composed of multiple membrane-bound and cytosolic subunits and when activated catalyzes the one-electron reduction of oxygen to superoxide. Superoxide and its highly reactive derivatives are essential for killing microorganisms. Rac proteins undergo posttranslational processing, primarily the addition of an isoprenyl group to a carboxyl-terminal cysteine residue. We directly compared recombinant Rac1 and Rac2 in a human neutrophil cell-free NADPH oxidase system in which cytosol was replaced by purified recombinant cytosolic components (p47-phox and p67-phox). Processed Rac1 and Rac2 were both highly active in this system and supported comparable rates of superoxide production. Under different cell-free conditions, however, in which suboptimal amounts of cytosol were present in the assay mixture, processed Rac2 worked much better than Rac1 at all but the lowest concentrations. This suggests that a factor in the cytosol may suppress the activity of Rac1 but not of Rac2. Unprocessed Rac proteins were only weakly able to support superoxide generation in either system, but preloading of Rac1 or Rac2 with guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) restored activity. These results indicate that processing is required for nucleotide exchange but not for interaction with oxidase components.
Mol Biol Cell 1993 Mar
PMID:Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase. 838 55

Superoxide is produced by phagocytic cells at rates sufficient to have cytocidal effects. A wide variety of receptor-dependent and -independent agonists triggers this respiratory burst, including immunoglobin aggregates, complement fragments, and leukotriene B4. Lower rates of O2-. production are triggered by addition of specific cytokines into B-lymphocytes, endothelial cells, fibroblasts, and kidney mesangial cells; low concentration of radicals may act as signals for proliferation or other changes. The NADPH oxidase of phagocytes, characterized by the presence of FAD and a low potential cytochrome b, is organized to transfer electrons electrogenically across the plasma membrane from NADPH to O2. A proton channel permits movement of compensating H+.
Mol Chem Neuropathol
PMID:The mechanism of the production of superoxide by phagocytes. 839 50


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