Inhibitors of NADPH oxidase as guides to its mechanism.
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Binding Sites↗
Biomedical subjects
O T Jones
Publications and source records attributed to O T Jones.
Differential effect of cyclosporine A on respiratory burst by several types of human leukocytic cells.
The effect of cyclosporine on PMA-stimulated superoxide production has been studied on human alveolar macrophages, human neutrophils, cytoplasts and Epstein-Barr-virus-transformed B lymphocytes. Cyclosporine inhibits superoxide production in alveolar macrophages but not in neutrophils and cytoplasts. The respiratory burst of B-lymphocytes was scarcely inhibited by cyclosporine. The activity of NADPH oxidase from macrophages and neutrophils was not directly affected by cyclosporine. These data are considered in relation with the proposed mechanism for cyclosporine action and the stimulation of the respiratory burst.
Cyclosporine↗
Distribution of Ca2+ channels on frog motor nerve terminals revealed by fluorescent omega-conotoxin.
Tetramethylrhodamine-conjugated omega-conotoxin was used as a fluorescent stain (Jones et al., 1989) to determine the spatial distribution of voltage-gated Ca2+ channels along frog motor nerve terminals. Like native omega-conotoxin, the fluorescent toxin blocked neuromuscular transmission irreversibly. The fluorescent staining was confined to the neuromuscular junction and consisted of a series of narrow bands (in face views) or dots (in side views) approximately 1 micron apart. This characteristic staining pattern was prevented by pretreatment with omega-conotoxin and by prior denervation for 5-7 d. Combined fluorescence and phase-contrast optics indicated that the stain was on the synaptic rather than the nonsynaptic side of the nerve terminal. The bands and dots of stain proved to be in spatial register with the postsynaptic junctional folds, as revealed by combined staining of ACh receptors. It is concluded that the voltage-gated Ca2+ channels on frog motor nerve terminals are concentrated at active zones. The findings are consistent with the suggestion (Heuser et al., 1974; Pumplin et al., 1981) that the large intramembraneous particles seen at freeze-fractured active zones are voltage-gated Ca2+ channels.
Animals↗
Involvement of an NAD(P)H oxidase as a pO2 sensor protein in the rat carotid body.
The rat carotid body tissue reveals a photometrically measurable haem signal with absorbance maxima at 560 nm, 518 nm and 425 nm, suggesting the presence of a b-type cytochrome; this was confirmed by pyridine haemochrome and CO spectra. The quantity of cytochrome b was estimated to be 310 pmol.mg of protein-1. This haem is capable of H2O2 formation, which can be inhibited by 10 microM-diphenyliodonium (DPI). The hypoxia-induced increase in nervous chemoreceptor discharge and the reduction of FAD and NAD(P)+ were also inhibited by DPI (10 microM). These results suggest that an oxidase such as the NAD(P)H oxidase of neutrophils may act as a pO2 sensor protein in the rat carotid body, probably inducing the pO2 chemoreceptor process by H2O2 formation.
Animals↗
Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons.
The major cytochrome in microsomal membrane preparations from developing seeds of safflower (Carthamus tinctorius, var High Linoleate), has a reduced-minus-oxidized difference spectrum characteristic of a b-type cytochrome, and was identified from its midpoint-potential (E'7.2) value as cytochrome b5. Cytochromes P-450 and P-420 were also present. The cytochrome b5 content of microsomal preparations from a number of oilseed species was found to be in the order of 200-300 pmol/mg of protein. The cytochrome b5 was reduced in the membrane preparations by NADH, demonstrating the presence of an NADH: cytochrome b5 reductase; NADPH was a less effective donor. Microsomal membranes catalysed the NAD(P)H-dependent conversion of radioactive oleate into linoleate, indicating acyl-CoA: lysophosphatidylcholine acyltransferase and 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) activity. Desaturation of oleate to linoleate was unaffected by CO, but inhibited by CN-. The addition of oleoyl-CoA to the NADH-reduced membranes resulted in the CN(-)-sensitive partial re-oxidation of cytochrome b5, indicating that electrons from NADH were transferred to the site of desaturation via this cytochrome. The delta 12-desaturase in safflower, therefore, is CN(-)-sensitive and appears to require cytochrome b5 and NADH: cytochrome b5 reductase for activity.
Cytochromes b5↗
The use of diphenylene iodonium, an inhibitor of NADPH oxidase, to investigate the antimicrobial action of human monocyte derived macrophages.
Diphenylene iodonium is an inhibitor of the respiratory burst-generating NADPH oxidase of phagocytes. The effect of this compound on human monocyte-derived macrophages and its usefulness in exploring the antimicrobial mechanisms of phagocytes was examined. 1 microM diphenylene iodonium inhibited hydrogen peroxide production by human macrophages and the activity of these cells against Toxoplasma gondii. At this concentration macrophage degranulation was unaffected.
Acetylglucosaminidase↗
Purification and some properties of the 45 kDa diphenylene iodonium-binding flavoprotein of neutrophil NADPH oxidase.
The 45 kDa diphenylene iodonium-binding flavoprotein of the human neutrophil superoxide-generating oxidase has been purified by affinity chromatography. The polypeptide was eluted from Blue Memsep or 2',5'-ADP-agarose columns with either NADP or low concentrations of the specific inhibitor diphenylene iodonium. The purified protein was shown to bind FAD at a ratio of 1.09 mol of FAD/mol of protein. The reconstituted flavoprotein had a fluorescence spectrum similar, but not identical, to that of free FAD. It had an isoelectric point of approx. 4.0. The reconstituted flavoprotein displayed no diaphorase activity towards a range of artificial electron acceptors. Polyclonal antibodies raised against the pure protein inhibited superoxide generation by solubilized oxidase in a dose-dependent manner, and inhibited superoxide generation when incubated with either cytosol or membrane fractions in a reconstituted system. These antibodies precipitated the 45 kDa polypeptide together with a haem-containing 23 kDa protein thought to be the small subunit of cytochrome b-245. Antibodies raised against cytochrome P-450 reductase also precipitated these two polypeptides. These results are consistent with the 45 kDa polypeptide being the flavoprotein of the neutrophil superoxide-generating oxidase.
Cell-Free System↗
Superoxide generation by EBV-transformed B lymphocytes. Activation by IL-1 beta, TNF-alpha and receptor independent stimuli.
The generation of superoxide by Epstein-Barr virus (EBV)-transformed human B lymphocytes can be stimulated by a range of compounds; receptor-dependent stimuli include tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and lipopolysaccharides (LPS), and independent stimuli include AlF3, A21387 and ionomycin. The stimuli suggest that the activation pathway for the lymphocyte oxidase is similar to that proposed for the neutrophil oxidase. Although the rate of superoxide production was lower than that by neutrophils, the respiratory burst was much prolonged. It is possible that this superoxide generation by lymphocytes may have a biological function.
B-Lymphocytes↗
Superoxide generation is inhibited by phospholipase A2 inhibitors. Role for phospholipase A2 in the activation of the NADPH oxidase.
The stimulation of O2.- generation by phorbol 12-myristate 13-acetate (PMA) in human neutrophil-derived cytoplasts was inhibited by a variety of phospholipase A2 inhibitors in a concentration-dependent manner. Inhibition was found to be independent of the order of addition of the inhibitor and PMA. The most potent inhibitor, RO 31-4639, inhibited O2.- generation with an IC50 value (concentration causing 50% inhibition) of 1.5 microM. The addition of either arachidonic acid or SDS, in the presence of the inhibitors, was able to restore O2.- generation. The results suggest that arachidonic acid, released by phospholipase A2, is necessary for both the activation and the maintenance of O2.- generation by the NADPH oxidase.
Acetophenones↗
Studies on the electron-transfer mechanism of the human neutrophil NADPH oxidase.
A superoxide-generating NADPH oxidase was solubilized from phorbol 12-myristate 13-acetate-activated human neutrophils with a mixture of sodium deoxycholate (0.125%, w/v) and Lubrol-PX (0.125%, v/v). The solubilized preparation contained FAD (577 pmol/mg of protein) and cytochrome b-245 (479 pmol/mg of protein) and produced 11.61 mol of O2-./s per mol of cytochrome b (340 nmol of O2-./min per mg of protein). On addition of NADPH, the cytochrome b-245 was reduced by 7.9% and the FAD by 38% in the aerobic steady state; NADH addition caused little steady-state reduction of cytochrome b and FAD. In this preparation, and several others, the measured rate of O2-. production correlated with the turnover of cytochrome b calculated from the extent of cytochrome b-245 reduction under aerobic conditions. Addition of diphenyleneiodonium abolished the reduction of both the FAD and cytochrome b-245 components and inhibited O2-. production. The haem ligand imidazole inhibited O2-. generation and cytochrome b reduction while permitting FAD reduction. These results support the suggestion that the human neutrophil NADPH oxidase has the electron-transport sequence: NADPH----FAD----cytochrome b-245----O2.
Cytochrome b Group↗
Properties of the superoxide-generating oxidase of B-lymphocyte cell lines. Determination of Michaelis parameters.
The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration.
B-Lymphocytes↗
Localization and mobility of omega-conotoxin-sensitive Ca2+ channels in hippocampal CA1 neurons.
Voltage-dependent Ca2+ channels (VDCCs) are modulators of synaptic plasticity, oscillatory behavior, and rhythmic firing in brain regions such as the hippocampus. The distribution and lateral mobility of VDCCs on CA1 hippocampal neurons have been determined with biologically active fluorescent and biotinylated derivatives of the selective probe omega-conotoxin in conjunction with circular dityndallism, digital fluorescence imaging, and photobleach recovery microscopy. On noninnervated cell bodies, VDCCs were found to be organized in multiple clusters, whereas after innervation the VDCCs were concentrated and immobilized at synaptic contact sites. On dendrites, VDCC distribution was punctate and was interrupted by extensive bare regions or abruptly terminated. More than 85% of the dendritic VDCCs were found to be immobile by fluorescence photobleach recovery. Thus, before synaptic contact, specific mechanisms target, segregate, and immobilize VDCCs to neuronal cell bodies and to specialized dendritic sites. Regulation of this distribution may be critical in determining the firing activity and integrative properties of hippocampal CA1 neurons.
Animals↗
Superoxide-dependent nitroblue tetrazolium reduction and expression of cytochrome b-245 components by human tonsillar B lymphocytes and B cell lines.
EBV-transformed B lymphocyte cell lines can generate superoxide, using an electron transport chain homologous, or even identical, to phagocytic NADPH-oxidase. We searched for normal, not virally transformed, B lymphocytes with analogous properties, using tonsils as the source of B cells. Unseparated tonsillar leukocytes contained cells capable of PMA-triggered superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT+ cells) well in excess of phagocytes (18.9 +/- 6.4% NBT+ cells with 1.3 +/- 0.9% granulocytes and 1.9 +/- 2.3% monocytes/macrophages, n = 8). NBT reduction was also inhibited by diphenylene iodonium, a selective inhibitor of phagocytic NADPH-oxidase. Cross-linking of surface Ig was equally effective as PMA in inducing NBT reduction among tonsillar leukocytes. NBT+ cells co-distributed with B cells on Percoll density gradients and were enriched among purified B cells obtained by SRBC rosetting twice and Sephadex G10 adherence (47.8 +/- 15.2% NBT+ cells among 90.5 +/- 5.5% B cells, 4.8 +/- 5.1% T cells, 1.2 +/- 0.77% monocytes/macrophages, and 0.73 +/- 0.6% granulocytes, n = 10). Further, mAb 7D5, directed against an extracellularly located epitope of the small subunit of cytochrome b-245 of phagocytes, stained the majority of tonsillar B cells (85 +/- 9.2% 7D5+ cells and 91.6 +/- 4.04% B cells, n = 3). Superoxide production, staining with 7D5 antibody, and expression of mRNA for the beta chain of cytochrome b-245 were further analyzed in cell lines. The EBV-BLCL F1 and the Burkitt lymphoma P3HR-1 both carried 7D5-detectable cytochrome b-245 Ag and expressed mRNA for the beta chain of the cytochrome b, both in similar amounts. However, only F1, not P3HR-1, was capable of PMA-triggered superoxide production. These data indicate that also normal nontransformed B lymphocytes possess the capacity to generate superoxide by a system apparently similar to phagocytic NADPH-oxidase, provisionally termed "B cell oxidase." Discrepancies observed in certain B cells and lines between expression of cytochrome b components and stimulus-induced superoxide production may be related to an absence or low level of other oxidase components or of the signal transduction mechanism. Conceivably, production of superoxide and derived reactive oxygen species by B cells may have cytotoxic, immunomodulatory, or mutagenic effects on the B cells themselves or on cells in their immediate vicinity.
Adult↗
Reconstitution of the nicotinic acetylcholine receptor using a lipid substitution technique.
The nicotinic acetylcholine receptor was purified by affinity chromatography in the presence of dioleoylphosphatidylcholine (DOPC). A method for replacing the DOPC with other lipids was developed by using detergent solubilization with a large excess of the new lipid followed by sucrose density gradient centrifugation in detergent-free buffers to separate receptor-lipid complexes from excess lipid and detergent. Homogenous complexes of defined lipid composition could be easily prepared and the efficiency of substitution was independent of lipid type. However, the functional properties of the resulting lipid complexes depended on the lipid composition.
Animals↗
Superoxide generation by the electrogenic NADPH oxidase of human neutrophils is limited by the movement of a compensating charge.
The ability of the NADPH oxidase of human neutrophil-derived cytoplasts to generate O2.-anions on the addition of phorbol 12-myristate 13-acetate is severely reduced in the presence of valinomycin and Zn2+ ions. The addition of NH4Cl or carbonyl cyanide m-chlorophenylhydrazone (K+ medium only) to these cytoplasts results in a restoration of O2.- generation. At an elevated pHo CCCP restores a greater rate of O2.- generation. Increasing the concentration of Zn2+ ions reduces the extent of the generation of O2.- on the addition of PMA. The restoration of O2.- generation by NH4Cl or CCCP requires the presence of valinomycin. The restoration of O2.- generation appears to be dependent on the movement of NH4+ ions or the anionic form of the uncoupler across the plasma membrane. The activity of the electrogenic NADPH oxidase of cytoplasts is limited by the movement of an ion to act as a compensator. The NADPH oxidase therefore exhibits respiratory control.
Ammonium Chloride↗
A minimum number of lipids are required to support the functional properties of the nicotinic acetylcholine receptor.
The detergent sodium cholate was used to both solubilize and partially delipidate the nicotinic acetylcholine receptor from Torpedo californica. Using both native membranes and reconstituted membranes, it is shown that the detergent to lipid molar ratio is the most important parameter in determining the effect of the detergent on the functional properties of the receptor. Receptor-lipid complexes were quantitatively separated from detergent and excess lipids by centrifugation through detergent-free sucrose gradients. The lipid to protein molar ratio of the complexes could be precisely controlled by adjusting the cholate and lipid concentrations of the starting membranes. Analyses of both ion influx activity and ligand binding revealed that a minimum of 45 lipids per receptor was required for stabilization of the receptor in a fully functional state. Progressive irreversible inactivation occurred as the lipid to protein mole ratio was decreased below 45, and complete inactivation occurred below a ratio of 20. The results are consistent with a functional requirement for a single shell of lipids around the perimeter of the receptor.
Allosteric Regulation↗
The effect of the NADPH oxidase inhibitor diphenyleneiodonium on aerobic and anaerobic microbicidal activities of human neutrophils.
NADPH-dependent superoxide production by intact human neutrophils is inhibited by DPI (diphenyleneiodonium), when stimulated by either FMLP (N-formylmethionyl-leucyl-phenylalanine) or PMA (phorbol 12-myristate 13-acetate). Addition of 10 microM-DPI abolished the reduction of both the FAD and the cytochrome b components of the NADPH oxidase. DPI inhibition of the oxidase was associated with defective aerobic killing of staphylococci by human neutrophils. Anaerobic killing, phagocytosis, chemotaxis and motility were relatively unaffected by 10 microM-DPI. Degranulation of the azurophil and specific granules, induced by the soluble stimuli FMLP or PMA, and by particulate stimuli was decreased by the presence of DPI. The above effects of DPI on human neutrophils are similar to those found in chronic granulomatous disease.
Aerobiosis↗
Internal pH changes associated with the activity of NADPH oxidase of human neutrophils. Further evidence for the presence of an H+ conducting channel.
The internal pH (pHi) of cytoplasts, derived from human neutrophils, falls 0.05 pH units upon activation of the superoxide-generating NADPH oxidase. The decrease in pHi is absent in diphenyleneiodonium-treated cytoplasts and therefore it is likely to arise directly from the activity of the oxidase. The addition of amiloride, to diminish the Na+/H+ exchanger, enhanced the extent of the internal acidification but not the initial rate. However the electroneutral Na+/H+ exchanger cannot be a contributor to H+ efflux to compensate for charge translocated by the oxidase. In the presence of Cd ions or valinomycin, phorbol-induced acidification of the cytosol was greatly increased, suggesting an inability to translocate the cytosolic H+ generated by an electrogenic oxidase. In the presence of both Cd and valinomycin the cytoplasts retained 0.8 H+ per O2-. generated. The rate of acidification of the external medium by stimulated cytoplasts is greatly reduced in the presence of Zn and valinomycin. Our results support the view that the plasma membrane of neutrophils contains Zn2+- or Cd2+-sensitive proton-conducting channels which maintain a stable membrane potential and pHi during the activity of the electrogenic NADPH oxidase.
Cadmium↗