Tamas Bajor

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.

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.

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

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

Abstract.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-γ level were increased mainly in rat cells while TNF-α 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-γ, TNF-α and MIP-2 productions were in good correlation with NOS II expression.

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

AbstractObjective 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.

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 various indolyl amino acid derivatives on arginase activity in macrophages

Summary.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 α-helix content was detected by circular dichroism together with a decrease in parallel pleated sheet and β-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.