Rat alveolar macrophages require NADPH for superoxide production in the respiratory burst. Effect of NADPH depletion by paraquat

Alveolar macrophages can be stimulated by concanavalin A to produce extracellular superoxide. Conflicting opinions exist, however, concerning the relative importance of the oxidation of either NADPH or NADH in the generation of (Formula: see text) by surface membrane-stimulated phagocytic cells. Alveolar macrophages were obtained from adult male rats by lavage with phosphate-buffered saline. Cells (approximately 10(6)/ml) were incubated in Krebs-Ringer phosphate 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and ferricytochrome c for 15 min at 37 degrees C before addition of concanavalin A. Release of (Formula: see text) was detected as the difference in cytochrome c reduction, followed at 550 nm, in the absence and presence of superoxide dismutase. Superoxide production by concanavalin A-stimulated alveolar macrophages was markedly increased in the presence of glucose but fructose, lactate, and pyruvate were without effect. Paraquat (methylviologen), an oxidation-reduction dye, significantly reduced concanavalin A-stimulated (Formula: see text) production when incubated at 1 mM with alveolar macrophages in the absence of glucose. The effect of paraquat was reversed by glucose, but fructose, lactate, and pyruvate could not reverse paraquat inhibition. Paraquat enhanced oxidation of NADPH (but not NADH) by cell supernatant and increased pentose phosphate shunt activity in resting macrophages, but did not affect mitochondrial respiration or ATP content of alveolar macrophages. These results suggest that paraquat is able to specifically deplete NADPH in alveolar macrophages while not affecting NADH or ATP. Our conclusion is that NADPH is essential for the production of (Formula: see text) by concanavalin A-stimulated alveolar macrophages.     

ADP stimulates the respiratory burst without activation of ERK and AKT in rat alveolar macrophages

Alveolar macrophages (AM) are the first line of defense against infection in the lungs. We previously showed that the production of superoxide and hydrogen peroxide, i.e., the respiratory burst, is stimulated by adenine nucleotides (ADP > ATP) in rat AM through signaling pathways involving calcium and protein kinase C. Here, we further show that ADP induces a rapid increase in the tyrosine phosphorylation of several proteins that was reduced by the tyrosine kinase inhibitor genistein, which also inhibited the respiratory burst. Interestingly, ADP did not trigger the activation of the mitogen-activated protein kinases ERK1 and ERK2, or that of protein kinase B/AKT, a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway. This is in contrast to another stimulus of the respiratory burst, zymosan-activated serum (ZAS), which activates both the ERK and PI3K pathways. Thus, this study demonstrates that the receptor for ADP in rat AM is not coupled to the ERK and AKT pathways and, that neither the ERK pathway nor AKT is essential to induce the activation of the NAPDH oxidase by ADP in rat AM while tyrosine kinases appeared to be required. The rate and amount of hydrogen peroxide released by the ADP-stimulated respiratory burst was similar to that produced by ZAS stimulation. The absence of ERK activation after ADP stimulation therefore suggests that hydrogen peroxide is not sufficient to activate the ERK pathway in rat AM. Nonetheless, as hydrogen peroxide was necessary for ERK activation by ZAS, this indicates that, in contrast to ADP, ZAS stimulates a pathway that is targeted by hydrogen peroxide and leads to ERK activation.     

Inhibition of production of LTB4 and chemotactic agent from rat alveolar macrophages treated with t-butyl hydroperoxide is independent of ATP depletion

In rat alveolar macrophages treated with 100 microM t-butyl hydroperoxide (tBOOH), leukotriene B4 (LTB4) synthesis was significantly lower than the basal level while levels of cyclooxygenase pathway products were increased. LTB4, 5,6-dihydroxyeicosatetraenoic acid (5,6-DiHETEs), and 5-hydroxyeicosatetraenoic acid (5-HETE) production in macrophages was significantly stimulated by 2 microM A23187, but this was suppressed 40% by simultaneous addition of 10 microM tBOOH and completely abolished by 100 microM tBOOH. Basal and A23187-stimulated macrophage production of chemotactic agents were similarly suppressed by addition of tBOOH; this effect paralleled depression of cellular LTB4 synthesis. In contrast to the significant depression of A23187-stimulated formation of 5-lipoxygenase products by 10 microM tBOOH, cellular adenosine triphosphate (ATP) was unchanged. Macrophages pretreated with KCN led to a 42% decline in ATP levels; however, LTB4, 5,6-DiHETEs, and 5-HETE production in response to A23187 was not suppressed. The results indicate that inhibition of 5-lipoxygenase pathway products in macrophages treated with tBOOH did not occur by depletion of cellular ATP levels.     

Cadmium-induced depression of the respiratory burst in mouse pulmonary alveolar macrophages, peritoneal macrophages and polymorphonuclear neutrophils.

No profound alteration in the resting O₂ consumption of mouse pulmonary alveolar macrophages, polymorphonuclear neutrophils or peritoneal macrophages incubated in media containing either cadmium chloride or cadmium acetate was observed. However, when heat-killed $\text{P. aeruginosa}$, opsonized in autologous serum, were added to the cell suspension a significant depression in the respiratory burst accompanying the phagocytic event was manifested. The suppression of the respiratory burst appeared to be related to the concentration of cadmium. The possible alteration in the relationship between macrophage microtubule assembly and endocytosis is discussed.     

Effects of liposomes of different phospholipid composition on the induction of respiratory burst in human blood monocytes and alveolar macrophages.

Liposomes made from phosphatidylcholine (PC) or PC-fatty acid ester mixtures have been shown to induce an activation of the respiratory burst in human blood monocytes and alveolar macrophages (AM). Incorporation of 1,2-diacylglycerol or arachidonic acid into PC liposomes significantly enhanced the effect. In the case of AM, the effect of PC liposomes was similar to those of phorbol 12-myristate 13-acetate (PMA) and the ionophore A 23187, while in monocytes, PMA and A 23187 induced a stronger respiratory burst than PC liposomes. In the presence of PMA, higher liposomal concentrations were required to produce the maximum activation of the respiratory burst in both types of cells.     

Effects of neonatal monosodium-L-glutamate treatment on rat alveolar macrophages

Treatment of neonatal rats with repeated doses of monosodium-L-glutamate resulted in changes of morphology and function of alveolar macrophages recovered from adult female rats. Numerous cellular lipid vacuoles and lamellar structures were observed in these alveolar macrophages under transmission electron microscopy. There was approximately 50% increase of cellular total lipid content measured by Oil-Red-O staining and colorimetric method. The yeast phagocytosis and intracellular killing ability, as well as the inhibitory effect on the growth of tumor cells were reduced in alveolar macrophages from neonatal MSG-treated rats.     

Effects of fibronectin on actin organization and respiratory burst activity in neutrophils,monocytes, and macrophages

Previous studies have shown that fibronectin (Fn) enhances phagocytosis and killing of antibody-coated bacteria by neutrophils and macrophages. In an attempt to understand the mechanism of this enhancement, we have investigated the effects of Fn on phagocytosis-related actin organization as well as respiratory burst activity in neutrophils, monocytes and culture-derived macrophages. Employing an NBD-phallacidin flow cytometric analysis of filamentous actin formation, we found that Fn promotes rapid actin polymerization within 30 seconds in neutrophils, monocytes, and macrophages, but not lymphocytes. Enhancement of actin polymerization by Fn was concentration-dependent and mediated by a pertussis toxin- but not cholera toxin- sensitive G protein. Inhibition of protein kinase C by sphingosine (20 μM), calcium influx by verapamil (0.1 mM), or intracellular calcium mobilization by 8-(N, N-diethyl-amino) octyl-3,4,5-trimethoxybenzoate HCI (TMB-8; 0.1 mM) did not block Fn-enhanced actin polymerization in phagocytes. Incubation of neutrophils and macrophages on microtiter plates precoated with Fn suppressed superoxide (O₂^?) production induced by IgG- and IgA- opsonized group B streptococci. In contrast, Fn significantly enhanced IgA- and IgG-mediated O₂^? production by freshly isolated monocytes. These data suggest that Fn enhances phagocytosis, presumably through G protein-coupled cytoskeleton reorganization and augments O₂^? production by circulating monocytes. In contrast, it appears to suppress O₂^? production by the active phagocytic cells, neutrophils and macrophages. This may result in enhanced phagocytosis and intracellular killing of microorganisms without damaging interstitial tissues. © 1994 Wiley-Liss, Inc.     

Respiratory burst in alveolar macrophages of diabetic rats

Bactericidal ability of alveolar macrophages is depressed in rats with diabetes mellitus. To define the mechanism of this abnormality, we measured the parameters of respiratory burst in alveolar macrophages, peripheral blood monocytes, and neutrophils of rats 8 wk after the induction of diabetes by streptozocin. Superoxide anion (O2-.) generation during basal conditions and after stimulation with phorbol myristate acetate (PMA) was measured as superoxide dismutase-inhibitable cytochrome c reduction. NADPH, the principal substrate for NADPH-oxidase-dependent O2-. generation, was measured in the alveolar macrophages and quick-frozen lungs by the enzyme-cycling method. O2-. generation after PMA was significantly lower in the alveolar macrophages of diabetics than in the controls (14.4 +/- 2.0 nmol.10(6) cells-1.20 min-1 vs. 26.2 +/- 1.9, P less than 0.05). Conversely the peripheral blood monocytes of diabetics demonstrated an enhanced O2-. production after PMA stimulation. There was no significant difference in the neutrophil O2-.-generation between the groups. The alveolar macrophage NADPH (control 0.44 +/- 0.15 nmol/10(6) cells vs. diabetic 0.21 +/- 0.04, P less than 0.05) and lung tissue NADPH levels (control 81.4 +/- 16.3 nmol/g dry wt vs. diabetic 35.8 +/- 20.5, P less than 0.05) were significantly lower in the diabetics than in the controls. These data indicate that the O2-.-generating capacity of alveolar macrophages is markedly depressed in diabetes, whereas their precursors, monocytes, are primed to generate O2-. with PMA stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effect of t-butyl hydroperoxide on mitochondrial oxidative phosphorylation in isolated rat hepatocytes

Using high-resolution oxygraphy, we tested the changes of various parameters characterizing the mitochondrial energy provision system that were induced by peroxidative damage. In the presence of succinate as respiratory substrate, 3 mM t-butyl hydroperoxide increased respiration in the absence of ADP, which indicated partial uncoupling of oxidative phosphorylation. Low activity of coupled respiration was still maintained as indicated by the ADP-activated and oligomycin-inhibited respiration. However, during the incubation the phosphorylative capacity decreased as indicated by the continuous decrease of the mitochondrial membrane potential. Under these experimental conditions the maximum capacity of the succinate oxidase system was inhibited by 50% in comparison with values obtained in the absence of t-butyl hydroperoxide. Our data thus indicate that the oxygraphic evaluation of mitochondrial function represents a useful tool for evaluation of changes participating in peroxidative damage of cell energy metabolism.     

Rooperol, an inhibitor of cytokine synthesis, decreases the respiratory burst in human and rat leukocytes and macrophages

Luminol-enhanced chemiluminescence was measured in fresh whole human blood, or human neutrophils isolated from heparinized blood, human alveolar macrophages and rat alveolar macrophages stimulated with bacterial endotoxin (LPS). Tetraacetate esters of rooperol, a dicatechol showing anticytokine activity, added to cells simultaneously with LPS inhibited the respiratory burst. The effective concentrations of rooperol were in the range of 1-10 muM depending on cell type and corresponded well with inhibition of nitric oxide production by rat alveolar macrophages. Thus rooperol may reduce some effects of excessive phagocytic activity and inflammatory reaction but by quenching free radicals production may also diminish the resistance to bacterial infections.     

Thymine hydroperoxide, a substrate for rat Se-dependent glutathione peroxidase and glutathione transferase isoenzymes

The thymine hydroperoxide, 5-hydroperoxymethyluracil, is a substrate for Se-dependent glutathione (GSH) peroxidase and the Se-independent GSH peroxidase activity associated with the GSH transferase fraction. These enzymes may contribute to repair mechanisms for damage caused by oxygen radicals. GSH transferases 1-1, 2-2, 3-3, 4-4, 6-6, and 7-7 [(1984) Biochem. Pharmacol. 33, 2539-2540] are shown to differ considerably in their ability to utilize this substrate. For example, high activity is found in GSH transferase 6-6 which is the major isoenzyme in spermatogenic tubules where DNA synthesis is so active and faithful DNA replication so important. The activity of the purified GSH transferase isoenzymes towards 5-hydroperoxymethyluracil is comparable with their activity towards other endogenous substrates related to cellular peroxidation such as linoleate hydroperoxide and 4-hydroxynon-2-enal or biologically important xenobiotic metabolites such as benzo(a)pyrene-7,8-diol-9,10-oxide.     

Effects of variation in glutathione peroxidase activity on DNA damage and cell survival in human cells exposed to hydrogen peroxide and t-butyl hydroperoxide.

The selenium-dependent glutathione peroxidase activities of two human cell lines, the colon carcinoma HT29 and the mesothelioma P31, cultured in medium containing 2% serum, increased from 195 to 541 and from 94 to 361 units/mg of protein respectively after supplementation with 100 nM-selenite. The catalase activity remained unchanged by this treatment. The effects of the obtained variation in glutathione peroxidase activities were investigated by exposing cells to H2O2 and t-butyl hydroperoxide. Selenite supplementation resulted in a decrease in H2O2-induced DNA single-strand breaks in both HT29 and P31 cells. A small, but significant, decrease in the number of DNA single-strand breaks for low doses (10-50 microM) of t-butyl hydroperoxide was found only in P31 cells and not in HT29 cells. We could detect neither induction of double-strand breaks (detection limit approx. 1000 breaks per cell) nor DNA-protein cross-links after exposing the cells to the two peroxides. In spite of the apparent protective effect of increased glutathione peroxidase activity on DNA single-strand break formation, there were no differences between selenite-supplemented and non-supplemented cells in cell survival after exposure to peroxide.     

Glutathione enzymes,glutathione content and t-butyl hydroperoxide induced lipid peroxidation in the gill and digestive gland of the estuarine clam,Rangia cuneata

1. Reduced glutathione (GSH), glutathione reductase (GSSG-reductase) and glutathione peroxidase (GSH-peroxidase) activities were measured in the gill and digestive gland of Rangia cuneata.2. Substantial GSH concentrations were found in both gill (820 ± 80 nmole/g tissue) and digestive gland (930 ± 130 nmole/g tissue). The digestive gland exhibited 2.5-fold greater GSSG-reductase activities and 0.5-fold lower GSH-peroxidase activities relative to the gill.3. In vivo exposure to t-butyl hydroperoxide (BHP) elicited a dose-dependent increase (P < 0.05) in lipid peroxidation in both tissues. Lipid peroxidation occurred earlier and to a greater extent in the digestive gland versus the gill. GSH concentrations in both tissues were unaffected by BHP exposure.4. The study results indicate that gill and digestive gland differ in susceptibility to BHP induced oxidative damage, and the difference is accounted for by differences in tissue GSH metabolism.     

Protective effect of ellagic acid on t-butyl hydroperoxide induced lipid peroxidation in isolated rat hepatocytes

Ellagic acid, a plant polyphenol, showed protective effect on isolated rat hepatocytes against destruction due to lipid peroxide formation induced by t-butyl hydroperoxide in vitro. Ellagic acid inhibited the generation of superoxide anions and hydroxyl radicals both in enzymic and non enzymic systems, thus providing protection against oxidative damage.     

Activation of several MAP kinases upon stimulation of rat alveolar macrophages: role of the NADPH oxidase.

Zymosan-activated serum (ZAS), a source of C5a, stimulates the rat alveolar macrophages (AM) to release superoxide anion. Here we show that treatment of rat AM with ZAS induced a time-dependent increase in the tyrosine phosphorylation of several proteins (116, 105-110, 82-78, 66-72, 62, 45, 42, and 38 kDa). This increase was sensitive to genistein, a tyrosine kinase inhibitor. ZAS stimulated the tyrosine phosphorylation and activation of three members of a family of serine/threonine kinases known as the mitogen-activated protein kinases (MAPK), i.e., ERK1 and ERK2, as assessed by immunoblotting, immunoprecipitation, and phosphotransferase activity, and p38 MAPK, as determined by immunoblotting with phospho-specific antibodies. In addition, ZAS induced the tyrosine phosphorylation of the SHC proteins and their association with GRB2, suggesting a role for this complex in the activation of the ERK pathway. Addition of extracellular catalase during ZAS stimulation significantly reduced the tyrosine phosphorylation response and the activation of ERK1 and ERK2 and their activator MEK1/2 while it did not affect that of p38 MAPK and MKK3/MKK6. Superoxide dismutase marginally increased the response to ZAS, supporting a role for hydrogen peroxide. In contrast to the results with AM, stimulation of human neutrophils with ZAS in the presence of catalase minimally altered the activation of ERK1 and ERK2. These data show that, in ZAS-stimulated rat AM, activation of the respiratory burst and production of hydrogen peroxide via superoxide dismutation are largely responsible for the activation of the ERK pathway through an upstream target.     

Molecular basis for the enhanced respiratory burst of activated macrophages

Macrophages elicited by injection of agents that produce inflammation or obtained from animals infected with intracellular parasites are primed so that they respond to phagocytosis or exposure to phorbol myristate acetate with a marked increase in the respiratory burst. This capacity to respond to stimulation with increased release of reactive oxygen metabolites appears to play an essential role in the increased microbicidal capability of activated macrophages. Macrophages can be primed for this capacity by incubation in vitro with bacterial products, proteases, or gamma interferon. The molecular basis for this priming is presently under investigation. An increase in the number or affinity of plasma membrane receptors does not appear to explain priming. Changes in one or more of the transduction events responsible for stimulus-response coupling might lead to more efficient stimulation or function of the enzyme responsible for the respiratory burst; these events are just beginning to be studied in macrophages. Priming can be explained at least in part by a modification of the respiratory burst enzyme such that it binds its substrate NADPH, the source of electrons for reduction of oxygen to superoxide anion, more efficiently. Understanding the molecular basis for priming of the respiratory burst might permit its eventual therapeutic manipulation.     

Inactivation of 2-oxoglutarate dehydrogenase in rat liver mitochondria by its substrate and t-butyl hydroperoxide

Ketoacid oxidation in rat liver mitochondria was very sensitive to t-butyl hydroperoxide (t-BuOOH). Furthermore, 2-oxoglutarate and pyruvate each enhanced t-BuOOH-induced oxidative stresses of mitochondria, such as oxidation of pyridine nucleotides and GSH, inhibition of respiration with the other NAD-linked substrates, and peroxidation of mitochondrial lipids. We provide evidence that the t-BuOOH and ketoacid-induced effects are due to the failure of supply of NADH by 2-oxoglutarate dehydrogenase, and report the inactivation of the dehydrogenase in mitochondria by simultaneous addition of 2-oxoglutarate and t-BuOOH. Using the purified enzyme, we confirmed that t-BuOOH-induced inactivation of 2-oxoglutarate dehydrogenase was enhanced by its substrate and thiamine pyrophosphate protected the dehydrogenase from the inactivation. In contrast, succinate-dependent oxidation of mitochondria was not only scarcely affected by t-BuOOH, but also succinate protected against inactivation of 2-oxoglutarate dehydrogenase by t-BuOOH in mitochondria.     

Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole

The effect of zinc on lipid peroxidation initiated by either ferric-nitrilotriacetate, t-butyl hydroperoxide, or 3-methylindole was studied using primary monolayer cultures of rat liver parenchymal cells. The malondialdehyde content of the cells and culture medium was used to estimate the extent of lipid peroxidation. As the zinc concentration of the culture medium was increased from 1 to 48 microM, peroxidation was diminished. Cellular zinc and metallothionein levels were proportionally increased by supplemental zinc. Zinc supplementation of the medium inhibited NADPH-cytochrome c reductase activity and stimulated glutathione peroxidase activity. The uptake of iron into the hepatocytes was significantly reduced as the level of zinc was raised, suggesting that zinc antagonizes uptake of chelated iron into isolated hepatocytes and in this way blocks iron-induced peroxidation. Furthermore, induction of metallothionein synthesis by zinc may contribute to the reduction in free radicals. Spectra from electron spin resonance studies, using phenylbutylnitrone as a spin-trapping reagent, demonstrated that free radical production was inversely related to the zinc concentration of the culture medium. Spin trap data suggest that metallothionein added to lysed cells in vitro decreases free radical production. Studies using the spin trap, 3,3,5,5-tetramethylpyrroline-N-oxide indicated that cumulatively the predominant radical present in the cultures was a phenyl radical with hydroperoxide or methylindole. Collectively, our data demonstrate that zinc inhibits free radical production and lipid peroxidation in cultured hepatocytes. The mode of action of zinc could occur via free radical scavenging by zinc-induced metallothionein and/or by processes related to cytochrome P-450 and glutathione peroxidase, since these were also found to be sensitive to zinc supplementation levels of the culture medium.     

Crystallographic analysis of the binding of NADPH, NADPH fragments, and NADPH analogues to glutathione reductase

The binding of the substrate NADPH as well as a number of fragments and derivatives of NADPH to glutathione reductase from human erythrocytes has been investigated by using X-ray crystallography. Crystals of the enzyme were soaked with the compounds of interest, and then the diffraction intensities were collected out to a resolution of 3 A. By use of phase information from the refined structure of the native enzyme in its oxidized state, electron density maps could be calculated. Difference Fourier electron density maps with coefficients Fsoak - Fnative showed that the ligands tested bound either at the functional NADPH binding site or not at all. Electron density maps with coefficients 2Fsoak - Fnative were used to estimate occupancies for various parts of the bound ligands. This revealed that all ligands except NADPH and NADH, which were fully bound, showed differential binding between the adenine end and the nicotinamide end of the molecule: The adenine end always bound with a higher occupancy than the nicotinamide end. Models were built for the protein-ligand complexes and subjected to restrained refinement at 3-A resolution. The mode of binding of NADPH, including the conformational changes of the protein, is described. NADH binding is clearly shown to involve a trapped inorganic phosphate at the position normally occupied by the 2'-phosphate of NADPH. A comparison of the binding of NADPH with the binding of the fragments and analogues provides a structural explanation for their relative binding affinities. In this respect, proper charge and hydrogen-bonding characteristics of buried parts of the ligand seem to be particularly important.