Macrophage NO Synthase Research Articles

The pathogenetic role of the nitric oxide system and its metabolites in the development and progression of chronic heart failure

Aim: to study the activity of eNOS, the concentration of L‑arginine and stable nitric oxide metabolites, as well as the degree of endothelium dependent vasodilation with 5‑minute compression test of the brachial artery in patients with chronic heart failure (CHF) of ischemic origin depending on L‑arginine administration in the treatment scheme.Material and methods. In the present study were included 303 patients (204 men and 99 women) observed about CHF ischemic genesis. Patients of the main group A+ (101 persons), in addition to the main treatment, took L‑arginine at a dose of 1 g 3 times per day in syrup (Tivortin), the rest of the patients (202 persons) were included in the comparison group А - . As a control group were examined 20 healthy volunteers. The metabolism of NO and endothelium-dependent vasodilatation were studied by determining the activity of eNOS, and concentration of NO2/NO3, determining the level of L‑arginine in the blood, assessment of endothelial function with a compression test. The endothelial function of vessels was judged according to the dopplerography of the brachial artery using the method of Solomon et al.Results. In patients taking arginine were observed additional positive effect on the arginine concentration in the peripheral blood (51,63% against 43,53% in group A, p<0.01). The distribution of the patients according to the status of LV systolic function showed that the effect of additional application of arginine is observed only in patients with low LVEF (69,63% vs 58,32% in the group with low LVEF, p<0.01). Also it was discovered that the use of arginine promotes a significantly greater increase in eNOS activity (11,53% against 4.76 per cent, p<0.001. In comparison with patients within the groups A+ and A —, classified according to the state of LV systolic function, it was found a significantly more pronounced effect of arginine in patients with normal systolic LV function.Conclusions. Patients with chronic heart failure (CHF) of ischemic origin, with low left ventricle ejection fraction (LV EF), a violation of NO metabolism is noted. Decrease of L‑arginine concentration leads to decrease in endothelial NO-synthase activity, and increase of macrophage NO-synthase activity leads to high concentration of nitric oxide and its stable metabolites in peripheral blood. Endothelial vasomotor dysfunction is associated with decrease of vasodilatation and even paradox vasoconstriction (in our study in 10 patients (3.3%)). Inclusion of L‑arginine to standard therapy of CHF gives additional positive effect on improvement of metabolism of nitric oxide and endothelial vasomotor function.

Translational repression of inducible NO synthase in macrophages by l-arginine depletion is not associated with an increased phosphorylation of eIF2α

In mouse inflammatory macrophages the cytokine-mediated expression of inducible nitric oxide synthase (iNOS) is regulated by the availability of the substrate l-arginine. Following arginine starvation the levels of iNOS mRNA remain unimpaired, whereas the translation of iNOS protein is strikingly downregulated. In the present study we addressed the question, whether arginine-deficient macrophages follow the canonical integrated stress response (ISR) that in other cell types depleted of amino acids was characterized by the accumulation of phosphorylated (i.e. inactive) eukaryotic translation initiation factor-2α (eIF2α), the attenuation of global protein synthesis and the induction of certain stress response target genes. Unexpectedly, resting as well as stimulated inflammatory macrophages constitutively exhibited high levels of phosphorylated eIF2α, which was not further increased upon l-arginine starvation. At the same time, macrophages deprived of l-arginine showed a significant upregulation of the mRNA levels of ISR genes. From these data we conclude that l-arginine deficiency blocks the translation of iNOS and elicits a stress response in macrophages, both of which, however, do not result from an enhanced phosphorylation of eIF2α. Alternative modes of translational repression of iNOS need to be considered.

Resistance and susceptibility to Marek’s disease: nitric oxide synthase/arginase activity balance

The metabolic NO pathway, catalyzed by the enzyme NO synthase in macrophages, is a key defense element against viruses and tumors. However, arginase is an other enzyme able to metabolize the substrate l-arginine, and the two enzymes are alternatively regulated by Th1 and Th2 cytokines in murine macrophages. Marek’s disease is characterized by strong immunosuppression and development of T-cell lymphomas in chickens. Inoculation of the very virulent strain of MDV RB-1B induced strong and long-lasting arginase macrophage-dependent activity, which was inhibited by l-norvaline in vitro, but induced low NO production in monocytes and splenocytes from highly susceptible B 13/B 13 chickens. By contrast, in B 21/B 21 chickens genetically resistant to tumor development, RB-1B induced a weak and transient increase in arginase activity and strong NO production. The vaccinal HVT strain did not induce any arginase activity in monocytes or splenocytes. Moreover, vaccination with HVT prevented tumor appearance after RB-1B challenge and increase in arginase activity, but favored NO production in susceptible chickens. Differential expression of NO synthase and arginase was modulated in chicken macrophages, with IFN-γ and LPS being strong inducers of both, depending on the type of macrophage, and TGF-β 1 and PGE 2 stimulating only arginase activity. This increase in arginase activity in macrophages from chickens inoculated with Marek’s disease virus might thus be due to a direct effect of the virus on macrophages, possibly through viral products, or to indirect effects on the cytokine balance.

Expression in yeast and purification of functional macrophage nitric oxide synthase. Evidence for cysteine-194 as iron proximal ligand.

Mouse macrophage NO-synthase (mNOS) was expressed in a unique yeast-based system by using a three-step procedure which allows yeast growth and NOS expression to be uncoupled. Despite cytotoxic effects related to mNOS expression, levels of catalytically active enzyme up to 0.5 mg of protein per 5 L of culture was obtained after purification. Its electrophoretic, spectroscopic [lambda max = 446 nm for its Fe(II)-CO complex], and catalytic properties were similar to those previously reported for mNOS purified from macrophages. Recombinant mNOS catalyzed the NADPH-dependent oxidation of L-arginine to citrulline (Km = 7 +/- 3 microM) as well as the reduction of cytochrome C by NADPH [Km = 34 +/- 8 microM and Vm = 25 +/- 5 mumol min-1 (mg of protein-1)]. Two mutants of mNOS in which Cys 194 was replaced with either serine or histidine were constructed and expressed in the same yeast strain at a level higher than that of the wild type protein, as they appear less toxic for the host. Both mutants exhibited electrophoretic properties and activities toward cytochrome C reduction identical to those of wild type NOS. However, they were unable to catalyze the oxidation of L-arginine to citrulline and did not appear to bind heme (no appearance of peaks around 400 and 446 nm for the resting enzyme and its CO complex, respectively, in visible spectroscopy). These data provide the first experimental evidence in favor of previous suggestions that Cys 194 was the proximal iron ligand of mouse mNOS.

Reconstitution of the second step in NO synthesis using the isolated oxygenase and reductase domains of macrophage NO synthase.

Inducible macrophage NO synthase (iNOS) is a homodimer of 130 kDa subunits. Trypsinolysis of iNOS inactivates its NO synthesis activity and cleaves the enzyme into a dimeric oxygenase fragment that contains heme, tetrahydrobiopterin, and the substrate binding site and a monomeric reductase fragment that contains FAD, FMN, calmodulin, and the binding site for NADPH [Ghosh, D. I., & Stuehr, D. H. (1995) Biochemistry 34, 801-807]. In this paper, we describe the reconstitution of NO synthesis activity utilizing the isolated oxygenase and reductase domains of iNOS. Mixing the domains at various ratios showed that NO was not produced from L-arginine but could be formed from the reaction intermediate N omega-hydroxy-L-arginine (L-NOHA). The apparent Km with L-NOHA in the reconstituted system was 100 microM versus 19 microM for native iNOS. D-NOHA was not a substrate. Maximum specific activity (per heme) occurred at an oxygenase to reductase molar ratio of 4:1, with higher ratios causing some inhibition. Reconstitution of activity was associated with electron transfer between the domain fragments and led to an incomplete reduction of the oxygenase domain heme iron. L-NOHA, but not L-arginine, increased NADPH consumption in the reconstituted system. Between 2.5 and 3.0 NADPH were consumed per NO formed from L-NOHA, considerably higher than the stoichiometry obtained with native iNOS (0.5 NADPH oxidized per NO formed), indicating an uncoupled electron transfer between the domain fragments. Thus, the isolated iNOS reductase and oxygenase domains each retain their separate catalytic functions but interact to catalyze only the second step of NO synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Subunit Dissociation and Unfolding of Macrophage NO Synthase: Relationship between Enzyme Structure, Prosthetic Group Binding, and Catalytic Function

Macrophage NO synthase is a homodimer of 130 kDa subunits. Each subunit contains an oxygenase domain that binds iron protoporphyrin IX (heme) and tetrahydrobiopterin (H4biopterin) and a reductase domain that binds FAD, FMN, and calmodulin (CaM) [Ghosh & Stuehr (1995) Biochemistry 34, 801-807]. We have studied the dissociation and unfolding reactions of dimeric iNOS in urea to learn how enzyme structure relates to catalysis and prosthetic group binding. The iNOS dimer dissociated between 0 and 2.5 M urea, and the subunits partially unfolded at 2.5 M urea and above. Dimer dissociation was accompanied by loss of NO synthesis activity and release of bound H4biopterin from the protein. However, the dissociated subunits maintained their cytochrome c and ferricyanide reductase activities and retained near stoichiometric quantities of bound heme. The subunit unfolding transition was accompanied by loss of reductase activities and partial loss of bound heme but retention of bound flavins and CaM. The heme iron in the dissociated subunits remained coordinated through axial cysteine thiolate ligation. Kinetic analysis of dimer dissociation showed that loss of NO synthesis correlated with a loss of heme Soret absorbance at 398 nm and an appearance of absorbance bands at 377 and 460 nm, which were attributed to DTT coordination to the sixth position of the heme iron to form a mixed bisthiolate complex. Subunits could reassociate into a dimer when incubated with L-arginine and H4biopterin. Dimer formation correlated with proportional recoveries of NO synthesis and heme Soret absorbance at 398 nm. Thus, dimeric iNOS undergoes separate dissociation and unfolding transitions in urea, and each transition is accompanied by a loss of a specific catalytic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage NO synthase: characterization of isolated oxygenase and reductase domains reveals a head-to-head subunit interaction.

Macrophage NO synthase (NOS) is a dimeric enzyme comprising two identical 130 kDa subunits and contains iron protoporphyrin IX (heme), tetrahydrobiopterin, FAD, FMN, and calmodulin. We have carried out limited proteolysis to locate the domains involved in prosthetic group binding and subunit interaction. Trypsin cleaved the subunits of dimeric macrophage NOS at a single locus, splitting the enzyme into two fragments whose denatured molecular masses were 56 and 74 kDa. The smaller fragments remained dimeric in their native form (112 kDa), contained heme and tetrahydrobiopterin, and could bind L-arginine, CO, or imidazole. In contrast, the larger fragments were monomeric in their native form, contained FAD, FMN, and CAM, and bound NADPH. Although neither purified fragment alone or in combination catalyzed NO synthesis from L-arginine, the flavin-containing fragment did catalyze cytochrome c reduction at a rate that was equivalent to that of native dimeric NOS. These results indicate that trypsin cuts macrophage NOS into two domains that can exist and function independently of one another. The domain that binds heme, H4biopterin, and substrate is also responsible for maintaining the NOS dimeric structure, while the domain containing FAD, FMN, and CAM is not required for subunit interaction. This suggests a structural model for macrophage NOS in which the subunits align in a head-to-head manner, with the oxygenase domains interacting to form a dimer and the reductase domains existing as independent extensions.

Cellular localization of inducible nitric oxide synthase in experimental endotoxic shock in the rat.

1. Endotoxin induces a shock-like syndrome with increased nitric oxide synthesis. To clarify the cellular source of NO in endotoxic shock we used immunohistochemistry and in situ hybridization to localize inducible NO synthase in rats given lipopolysaccharide or Corynebacterium parvum and lipopolysaccharide. Immunohistochemistry was carried out with an antibody raised against a synthetic peptide of mouse macrophage NO synthase. In situ hybridization was performed with 35S-labelled oligonucleotide probes corresponding to cDNA sequences common to mouse macrophage inducible NO synthase and rat vascular smooth inducible NO synthase. Monocytes and macrophages were identified by immunohistochemistry with the mouse monoclonal antibody ED1. 2. After lipopolysaccharide alone, the major site of NO synthase induction was monocytes and macrophages in multiple organs, principally liver and spleen. Bronchial, bile duct, intestinal and bladder epithelium and some hepatocytes also expressed inducible NO synthase. Expression peaked at 5 h and had returned to normal by 12 h except in spleen. 3. After priming with C. parvum, lipopolysaccharide led to a similar distribution of inducible NO synthase as lipopolysaccharide alone, but in addition there was more prominent hepatocyte staining, staining in macrophage granulomas in the liver and inducible NO synthase was present in some endothelial cells in the aorta. 4. These findings provide a direct demonstration of the cellular localization of inducible NO synthase after lipopolysaccharide.

Diethyldithiocarbamate inhibits induction of macrophage NO synthase

We investigated whether sodium diethyldithiocarbamate (DETC), an inhibitor of the nuclear transcription factor kappa B (NFkappaB), modulates induction of NO synthase (NOS) in murine bone marrow-derived macrophages. A short exposure (between 1 and 16 h) of L929-cell medium-preconditioned macrophages to E. coli lipopolysaccharide (LPS) significantly increased the level of NOS mRNA, and elicited NO formation as detected by electron spin resonance spectroscopy and by the release of nitrite. DETC (0.1–1 mM) present during stimulation with LPS prevented the increase in NOS mRNA and the expression of NOS activity. These findings suggest that NFkappaB is involved in the signal transduction pathway linking stimulation of macrophages by LPS with transcription of, the gene encoding inducible NOS.

Suppression of low dose streptozotocin induced diabetes in mice by administration of a nitric oxide synthase inhibitor

Nitric oxide has recently been identified as the primary toxic effector molecule in the lysis of islet cells by inflammatory macrophages. We show here that N-nitro-L-argininemethylester (NAME), an inhibitor of endothelial and macrophage NO synthase partially suppresses diabetes development in the low dose streptozotocin induced diabetes model in C57BL/6J mice. Mean blood glucose levels were lower in the group receiving NAME throughout the observation periof of 30d (p < 0.05–0.001). Similar concentrations of NAME as expected in vivo were tested in vitro in macrophage — islet cell cocultures and were found to partially suppress NO production and islet cell lysis. We conclude that NO synthase activity is a pathogenetic factor in diabetes development.