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.

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.

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.

A lymphokine-like factor isolated by deoxycholate from the membrane of human lymphocytes.

1. Membranous protein fractions containing carbohydrates were solubilized from human tonsillar lymphocytes in the presence or basence of deoxycholate. The optimal conditions for the detergent treatment, high solubilized protein yield without cell disruption, temperature and time of the treatment and concentration of the detergent were elucidated. 2. The protein fractions inhibited both the mitogen-activated and the nonactivated DNA synthesis in human lymphocyte targets in vitro but did not affect the uptake of [3H]thymidine. The fractions had a slight effect on the amino acid incorporation into proteins and failed to influence the uptake of amino acids. 3. It is assumed that the investigated membranous proteins are lymphokine-like materials produced continuously by lymphocytes in vivo and are incorporated into the membrane of the cells.

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.

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.

Effect of emetine and chloroquine on phagocytic processes of rat macrophages.

Emetine and chloroquine caused a dose-dependent and time-dependent inhibition of bacterial phagocytosis in rat peritoneal macrophages. The effect of emetine was found to be stronger: 50% inhibition was achieved at 5 X 10(-6) M concentration. The inhibition of phagocytosis was irreversible. Neither the binding of bacteria to the surface of phagocytes nor membrane marker 5' nucleotidase was touched by this treatment. The adherence of macrophages to plastic dishes was also impaired by the drugs but only in a smaller extent. Both emetine and chloroquine blocked the amino acid incorporation into macrophage proteins. The effect of emetine was more marked; 10(-6) M decreased the incorporation to 25% of control, while 10(-4) M chloroquine produced a similar inhibition. The block of protein synthesis was also irreversible.

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.

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.

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.

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.

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.

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 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.

Effect of released lymphocyte proteins on human lymphocytes in vitro.

The effect on lymphocytes and Hep2 target cells of proteins obtained from the medium of human lymphocytes incubated for short times was studied. The supernatants were precipitated and fractionated with ammonium sulfate in three steps. Two protein fractions were found to inhibit protein synthesis in target cells and to increase in vitro release of chromium from prelabelled lymphocytes. The proteins studied may be related to lymphotoxin produced by mitogen-activated lymphocytes. It is assumed that in vitro interactions for short times between different types of lymphocytes may cause the release of proteins. The same proteins play probably a part in the in vivo regulation of the metabolic activity of lymphocytes.