Characterization of oxidation of glutathione by cytochrome c.

Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) cytochrome c can be reduced by gluthatione as well as ascorbic acid. We investigated the effect of pH, Ca2+, Mg2+ and anionic phospholipids on the reduction of cytochrome c by glutathione.The reduction of cytochrome c by thiols was measured using photometry. Mitochondrial oxygen consumption was detected by use of oxygen electrode. Glutathione does not reduce cytochrome c at pH = 7.0 in the absence of Ca2+ and Mg2+. The reduction of cytochrome c by glutathione is inhibited by anionic lipids, especially cardiolipin. The typical conditions of apoptosis-elevated pH, Ca2+ level and Mg2+-increases the reduction of cytochrome c. Glutathione (5 mM) causes increased mitochondrial O2 consumption at pH = 8.0, in the presence of ADP either 1 mM Mg2+ or 1 mM Ca2+. Our results suggest that membrane bound cyt c does not oxidize glutathione. Free (not membrane bound) cytochrome c can oxidize glutathione. In mitochondria, O2 is depleted only in the presence of ADP, so the O2 depletion observed in the presence of glutathione can be related to the respiratory chain. Decreased glutathione levels play a role in apoptosis. Therefore, membrane unbound cyt c can contribute to apoptosis by oxidation of glutathione.

17-ß-estradiol Decreases Neutrophil Superoxide Production through Rac1.

Neutrophil granulocytes form the biggest free radical producing system of the human body. The importance of this system in atherosclerotic plaque formation and other free radical mediated disorders is confirmed by both in vivo and in vitro studies. Estrogen's effect on free radical production involves multiple estrogen receptors and occurs both on transcriptional and on protein phosphorylational level. Estrogen decreases the superoxide production of neutrophil granulocytes in such a short time frame it is unlikely to be mediated by transcription regulation. We investigated the underlying mechanism through which the mentioned estrogen effect takes place using an immunabsorption-based method. Phosphorylation data of 43 different messenger proteins were used for pathway analysis. The newly identified pathway involved largely second messengers from previously described non-genomic estrogen effects and affected superoxide production via Rac1 - an important regulator of free radical production and chemotaxis. Selective inhibition of the participating second messengers altered superoxide production in the predicted direction confirming that this pathway is at least partly responsible for the effect of 17-ß-estradiol on chemoattractant induced superoxide production.

The effect of various inflammatory agents on the phagocytosis and cytokine profile of mouse and rat macrophages.

OBJECTIVE AND DESIGN: The effects of various inflammatory stimuli on the cytokine profile and phagocytic capacity of mouse and rat peritoneal macrophages were investigated in vitro. The correlations between cytokine concentrations and the expressions of NOS II and arginase were also studied. METHODS: Mice and rats were injected intraperitoneally with various inflammatory agents. Peritoneal macrophages were isolated. The levels of eight cytokines were determined in macrophage cultures by ELISA test. Phagocytic capacity of macrophages was measured by the ingestion of M. Luteus. RESULTS: The most marked changes caused by i. p. treatments were observed in the levels of IL-1 and IL-6 in mice and of IL-12 in rats. IFN-gamma level were increased mainly in rat cells while TNF-alpha production was rather enhanced in mice. Phagocytic capacity of macrophages was higher in rat samples and it increased with all treatments, except BCG, without marked differences between different treatments. CONCLUSIONS: Each inflammatory agent caused an increase in cytokine productions in both species, with marked differences among cytokines. Correlations were found in mouse between IL-6 level and NOS II expression, and IL-10 level with arginase expression. In rat macrophages, IFN-gamma, TNF-alpha and MIP-2 productions were in good correlation with NOS II expression.

The inhibitory effect of various indolyl amino acid derivatives on arginase activity in macrophages.

Numerous indolyl amino acids and their derivatives inhibited arginase activity. The inhibition was found to be non-competitive, - at least partly - allosteric, and independent on manganese ions in the active site, and it cannot be explained by the dissociation of arginase homotrimers. Indole alone is weakly inhibitory; however, the presence of three-carbon side chains and their net charges is favorable for the inhibition. The binding of the inhibitory compounds caused only minor changes in the steric structure of arginase: a slight increase in alpha-helix content was detected by circular dichroism together with a decrease in parallel pleated sheet and beta-turn sections. A slight alteration in the tertiary structure was also found using tryptophane fluorescence studies, but buried apolar side chains were not transposed to the protein surface. Computer studies that were performed did not provide additional structural information.

The effect of various inflammatory agents on the alternative metabolic pathways of arginine in mouse and rat macrophages.

OBJECTIVE AND DESIGN: The effects of various inflammatory stimuli on the alternative arginine metabolic pathways in mouse and rat peritoneal macrophages were investigated in vitro and compared. TREATMENTS: Mice and rats were injected i. p. with thioglycollate, carrageenan, casein, BCG and Newcastle Disease Virus (NDV) vaccines. METHODS: Peritoneal macrophages were isolated from untreated and treated animals. The activities of nitric oxide synthase (NOS) II and arginase were measured and expressions were followed by Western blotting. The uptake of arginine and nitrite formation of macrophages were also measured. RESULTS: Inflammatory stimuli increased the NO production and the expression and activity of both NOS II and arginase in mice in vitro. On the contrary, the same treatments changed the expression and activity of NOS II only, but not those of arginase in rats. The most marked effects on NO metabolism were produced by casein and NDV treatments. CONCLUSIONS: The activity and expression of NOS II and arginase can be stimulated in peritoneal macrophages in vitro by injecting inflammatory agents into the peritoneal cavity. A markedly different response in arginine metabolism was observed in mouse and rat macrophages. Casein treatment was a potent inducer for both enzymes. NDV vaccines induced mainly NOS II, while thioglycollate induced arginase.

The inhibition of retinal inducible nitric oxide synthase overexpression and the attenuation of experimental uveitis by anti-inflammatory drugs in rats.

OBJECTIVE AND DESIGN: The effect of a steroid and a non-steroid anti-inflammatory drug on the inducible nitric oxide synthase (NOS II) in rats suffering from lipopolysaccharide (LPS)-induced uveoretinitis was studied. TREATMENTS: Rats were injected with LPS to induce uveitis and divided into three groups: treated with LPS only, LPS + dexamethasone and LPS + indomethacin, respectively. METHODS: Retinal, peritoneal macrophages and white blood cells were isolated. The activity and the expression of NOS II were followed by citrulline formation and Western blotting, respectively. Phagocytosis of bacteria was also measured. RESULTS: The best induction of NOS II was achieved by the intravitreal administration of LPS. Dexamethasone and indomethacin significantly decreased the activity and the expression of inducible nitric oxide synthase in retinal tissue, peritoneal macrophages and white blood cells. LPS treatment also increased phagocytosis and neither dexamethasone nor indomethacin reversed this effect. CONCLUSIONS: The beneficial effects of these drugs in experimental uveitis are mediated, at least partly, by their inhibitory effect on NOS II induction.

Systematic investigation of different steroid precursors with respect to their effect on superoxide anion production by human neutrophil granulocytes.

Free radicals are involved in several pathological processes in living organisms, for example in athero- and oncogenesis. Some steroids are known to be effective antioxidants, while others do not play any such role. The aim of our study was to examine the antioxidant capability of different metabolites in the synthesis of steroid hormones. As a model, we chose human neutrophils producing superoxide anion, which is the source of many other radicals. Neutrophils were separated from healthy volunteers. Isolated cells were incubated with varying concentrations of steroid compounds and stimulated with N-formyl-Met-Leu-Phe. Superoxide anion production was determined by photometry. Neutrophils incubated with corticosterone and 18-hydroxy-deoxycorticosterone showed a significant reduction in superoxide production, whereas we found a significant enhancement in the presence of 11beta-hydroxyprogesterone. Furthermore, we observed a non-significant decreasing trend after incubation with cholesterol 3-sulphate and an increasing tendency using 11-hydroxyandrostenedione. We were also able to produce newer morphological and functional evidence of the role of myeloperoxidase enzyme in the steroidal antioxidant effect by electronic microscopy and use of sodium hypochlorite in our incubation model. Based on these results, we conclude that not only steroid end products but also their intermediate metabolites, most of which are also present in human plasma, partly influence free radical metabolism. Thus, this study provides further argument for the search for the molecular basis responsible for the antioxidant effect of steroid structures. This may lead to new opportunities for finding really efficient antioxidants, which might perhaps be used in a combined manner with other agents in the fight against certain life-threatening diseases.

Plasma concentration of myeloperoxidase enzyme in pre- and post-climacterial people: related superoxide anion generation.

Neutrophil granulocytes are involved in the pathogenesis of atherosclerosis also through their free radical generation. The aim of the study was to test how extracellular levels of myeloperoxidase (MPO; a granulocyte enzyme playing role in free radical production) change by age and what effect this change has on the production of the free radical superoxide anion by neutrophils. We also wanted to examine whether the antioxidant effect of different steroid hormones is realized through the MPO. Plasma myeloperoxidase concentrations of healthy blood donors were quantified by ELISA. Superoxide anion production was measured by photometry. Myeloperoxidase concentration was significantly lower in plasmas obtained from older women and men than in those from younger subjects. Adding the MPO inhibitors 4-aminobenzoic acid hydrazide (ABAH) and indomethacin to the granulocytes, the generation of superoxide anion increased and the decreasing effect of the steroids on superoxide production was inhibited. Incubating the neutrophils with the product of the reaction catalyzed by MPO itself (hypochlorite anion), we found significant decrease in superoxide generation. According to our results MPO seems to diminish the production of superoxide anion and so probably has an antioxidant ability. Therefore, its lower plasma levels may contribute to the increasing incidence of atherosclerosis and other free radical mediated disorders in old people. Thus, after further studies MPO might become one of the indicators of cardiovascular risk and the scavenger capacity in general.

Time course of inducible nitric oxide synthase activity following endotoxin administration in dogs.

An increased production of nitric oxide (NO) via the inducible isoform of NO synthase (iNOS) has been incriminated in the pathogenesis of septic shock. Since the time course of iNOS activity is not known during endotoxic shock in dogs, we measured iNOS activity, estimated by the rate of conversion of (14)C-arginine to (14)C-citrulline in the absence of calcium, in the heart, lung, liver, kidney, and gut at 1, 2, 3, 4, and 6 h after a bolus of Escherichia coli endotoxin (2 mg/kg, iv), in the dog. This model, including generous fluid administration, is associated with typical features of human septic shock, including low systemic vascular resistance, altered myocardial function and limited oxygen extraction capability. An increase in iNOS activity was observed at 4 h in the liver (0.24 vs 0.04 mU/mg/min) and at 6 h in the heart (0.26 vs 0.09 mU/mg/min). These findings may contribute to a better delineation of the involvement of NO in endotoxic shock, and to the evaluation of the therapeutic effects of NO inhibitors.

Indomethacin prevents the induction of inducible nitric oxide synthase in murine peritoneal macrophages and decreases their nitric oxide production.

Indomethacin (0.14-.5 mM concentration) inhibits nitric oxide production in murine peritoneal macrophages. This was evidenced by measuring both nitrite production or 14C-L-citrulline formation. The inhibition was caused by the diminution of de novo inducible nitric oxide synthase production as demonstrated by Western blotting experiment. The effect of indomethacin after 4 h treatment was irreversible. NO synthase and arginase activities and the uptake of arginine were not directly affected by the drug. Indomethacin also decreased uridine incorporation in macrophages. The effect of indomethacin on the induction of other enzymes (i.e. arginase) was weaker.

Apoptosis inhibitory effect of the isothiourea compound, tri-(2-thioureido-S-ethyl)-amine.

Sulfhydril compounds, some of them with immunomodulatory activity have also been shown to modulate the induction of apoptosis. This study was performed to assess the possible apoptosis inhibitory effect of the immunomodulatory compound tri-(2-thioureido-S-ethyl)-amine (K-1) in U-937 and HL-60 leukaemia cell lines as model systems. Treatment of U-937 and HL-60 cells with K-1 inhibited etoposide (ETO)-induced apoptosis in both cell lines in a dose-dependent manner, IC(50)=200 microg/ml. The results indicate that inhibition of ETO-induced apoptosis occurs downstream of ETO-mediated cleavable complex formation but early in, or at the level of the mitochondria events of the apoptotic pathway. Further, like other isothioureas, K-1 proved to be a potent inhibitor of nitric oxide synthase. This raises the possibility that where the activation of nitric oxide synthase is involved in apoptosis induction, K-1 might also be effective. These findings suggest that K-1 may serve as a potent inhibitor of apoptosis initiated by ETO or nitric oxide.

Action of chloroquine on nitric oxide production and parasite killing by macrophages.

Chloroquine is known to inhibit several functions of macrophages, but its effect on the nitric oxide (NO)-dependent parasite killing capacity of macrophages has not been documented. NO synthesis by interferon-gamma-induced mouse and casein-elicited rat macrophages was significantly and irreversibly inhibited by chloroquine. The activity of the inducible NO synthase was not directly altered, but previous incubation of macrophages with chloroquine decreased it. Chloroquine did not alter arginase activity or arginine uptake. NADPH diaphorase activity, an indicator of NO synthase was impaired. Western blotting showed that inducible NO synthase synthesis was blocked by chloroquine. The blocking of NO formation by chloroquine resulted in increased infection of mouse peritoneal macrophages by Trypanosoma cruzi (T. cruzi). This suggests that chloroquine decreases NO formation by macrophages by inhibiting the induction of NO synthase. The findings are further evidence that NO is involved in the anti-parasitic response of macrophages.

Comparison of the inhibitory effect of isothiourea and mercapto-alkylguanidine derivatives on the alternative pathways of arginine metabolism in macrophages.

Novel, non-arginine based compounds have been identified as potent inhibitors of nitric oxide synthase (NOS). Members of the isothiourea and mercapto-alkylguanidine classes have generated much interest, as some members of these classes show selectivity towards the inducible isoform of NOS (iNOS), which plays a role in inflammation and shock. Here we compared the effect of a number of these compounds as well as L-arginine based NOS inhibitor reference compounds on macrophage-derived and liver arginase and macrophage iNOS activities. From the non-arginine based NOS inhibitors studied only S-aminoethyl-isothiourea (AETU) caused a slight inhibition of arginase activity. This inhibition was kinetically competitive and due to the rearrangement of AETU to mercapto-ethylguanidine (MEG). The weak inhibitory effect of non-arginine based iNOS inhibitors on arginase activity further supports the view that such compounds may be of practical use for inhibition of NO production in cells simultaneously expressing iNOS and arginase.

The inhibitory effect of nitrite, a stable product of nitric oxide (NO) formation, on arginase.

Macrophages contain arginase and an inducible NO synthase, demonstrated by using L-arginine, the common substrate, for production of both nitric oxide and urea. Arginase was inhibited by nitrite, the stable end product of NO. This inhibition was non-competitive, and could not be explained by the reaction of nitrite with arginine, or by the irreversible covalent modification of arginase, or by the removal of Mn2+, a cofactor of arginase.

Computer-aided comparison of the inhibition of arginase and nitric oxide synthase in macrophages by amino acids not related to arginine.

Macrophages contain arginase and an inducible nitric oxide (NO) synthase that use the same substrate, L-arginine, to produce nitric oxide and urea, respectively. Arginase was inhibited by various amino acids not related to L-arginine. These compounds were bound to the substrate binding site of the enzyme as supported by kinetic studies. Five binding sites were defined in this area by computer-aided analysis, and three complementary sites in a compound were sufficient to give an inhibitory character. NO synthase could not be inhibited by these compounds, but certain derivatives (e.g., putrescine or L-valinol) caused a marked and probably allosteric inhibition. The possible biological importance of these inhibitions in the tumoricid function of macrophages is discussed.

Comparison of substrate and inhibitor specificity of arginase and nitric oxide (NO) synthase for arginine analogues and related compounds in murine and rat macrophages.

Arginine utilizing enzymes in macrophages showed different specificities for various arginine analogues and derivatives as substrates and inhibitors. Isolated arginase was strongly inhibited by L-canavanine(Can) and L-ornithine(Orn) but only slightly by L-homoarginine(Hom) and L-argininamide(ArgNH2). These effects were not or only weakly observed when released urea was measured in long term cell cultures. On the other hand, both L-canavanine and L-argininamide were substrates for arginase in long-term cultures. The known inhibitors of NO synthase were ineffective. The mechanisms of inhibition were different for L-canavanine and L-ornithine, but clear mechanisms could not be identified). NO synthase was studied only in long term cell cultures without purification. Certain N-guanidino (NG)-substituted arginine derivatives caused a marked inhibition while inhibitors of arginase had only slight or no effect. L-homoarginine was also found to be the substrate of NO synthase. The comparison of these effects of arginine analogues and derivatives made possible a computer-aided approximation for the fitting of active centers of these enzymes to their substrates.

Arginine supply for nitric oxide synthesis and arginase is mainly exogenous in elicited murine and rat macrophages.

L-arginine, the precursor of nitric oxide(NO) is provided mainly by extracellular sources in casein-elicited murine and rat peritoneal macrophages. Free extracellular L-arginine(Arg), esters, peptides and proteins containing Arg are the best sources in accordance with the fact that proteolytic activity is high in peritoneal macrophages. The recycling of Arg from citrulline(Cit) was observed but at a low rate. This situation is different from that in endothelial cells where half of Arg is recycled from citrulline. No significant anaplerotic reaction from glutamic acid(Glu) can be demonstrated in peritoneal macrophages.

The salvage of deoxycytidine into dCDP-diacylglycerol by macrophages and lymphocytes.

Extracellular deoxycytidine (CdR) was previously shown to be salvaged into water soluble [1] and also into lipidic [2] precursors of phospholipids in stimulated lymphocytes and in lymphoma cells [3]. In this paper we have described that non-dividing murine macrophages salvaged not only 5-3H-CdR but also tritiated thymidine (3H-TdR) mainly into the pools as nucleotides. Chlorpromazine shifted the CdR salvage into a lipidic compound of the cells which was identified as 3H-dCDP-diacylglycerol (dCDP-DAG). After 5-3H-CdR labeling the lipid/DNA ratio was eleven times higher in macrophages than in tonsillar lymphocytes. Thin layer chromatography (TLC) on borate impregnated silica gel plates gave clear separation of CDP-DAG from dCDP-DAG supporting that the extracellular precursor for it is exclusively deoxycytidine and not ribocytidine. No interconversion between deoxy- and and ribocytidine could be observed neither in lymphocytes nor in macrophages.

Evidence for the pre-synthesized state of secreted macrophage arginase: arginase activity cannot be modified in short-term cultures.

The arginase produced by peritoneal macrophages is not synthesized de novo in short-term (3 h) cultures after harvesting the cells. In long-term cultures the arginase synthesis is restored. In contrast to arginase lysozyme is continuously synthesized in short-term cultures. These statements were proved by the following experimental results: 1. Protein synthesis inhibitor and lysosomotropic agents did not alter the arginase level. 2. Arginine and its analogue, canavanine and ornithine were not able to change the arginase activity. 3. The product of an alternative metabolic pathway of arginine, sodium nitrite, did not affect arginase activity. 4. Effectors influencing the synthesis of cyclic nucleotides (cAMP, cGMP), indomethacin, sodium nitroprusside and an analogue of cAMP had no effect on the arginase activity. 5. Arginase activity could not be significantly modified either by an in vitro Micrococcus luteus treatment or by changing the adherence period of peritoneal exudate cells. 6. When arginase was produced in murine peritoneal macrophages at various periods with medium change, the total arginase released into the media from murine and rat macrophages did not exceed the original intracellular arginase content of the adhered cells during the first 6 hours.

Inverse relation in the de novo arginase synthesis and nitric oxide production in murine and rat peritoneal macrophages in long-term cultures in vitro.

1. The de novo synthesis of arginase was much higher in murine than in rat peritoneal macrophages. This process was inhibited irreversibly by protein synthesis inhibitors and reversibly by glycolysis blockers. 2. Rat macrophages produce more nitric oxide (NO) than murine cells. NO production was inhibited by the inhibitors of protein synthesis or glycolysis. 3. The loading of macrophages by exogenous arginine for 24 hr in vitro resulted in the increase of arginase and nitrite in macrophages to different extents. 4. No great differences in lysozyme production was observed. 5. The proportion of arginine taken up and incorporated is contrasted in murine and rat macrophages.