Murine Macrophage Nitric Oxide Synthase Research Articles

Evaluation of Nitric Oxide Synthase Activity and Inhibition Kinetics by Chemiluminescence

The binding affinity (KI) and inactivation rate (kinact) parameters of nitric oxide synthase (NOS) inhibitors are typically estimated by kinetic activity studies. Methods currently used in the estimation of these parameters frequently employ radiolabeled materials and require intensive sample preparation. We have devised a simple, reproducible, and sensitive method for the kinetic analysis of NOS activity and inhibition kinetics using chemiluminescence. We have used this method to characterize enzyme activity for purified murine macrophage nitric oxide synthase (NOS II). Using this method, we have also estimated the inhibitory parameters for a series of competitive antagonists and mechanism-based inactivators of NOS II. The estimated parameters are in agreement with those reported using other methods. We conclude that the chemiluminescence method can be used for kinetic studies of NOS activity and inhibition. This method represents a more efficient means for conducting kinetic studies of NOS inhibition.

Reactions catalyzed by tetrahydrobiopterin-free nitric oxide synthase.

Murine macrophage nitric oxide synthase (NOS) was expressed in E. coli and purified in the presence (holoNOS) or absence (H4B-free NOS) of (6R)-tetrahydro-L-biopterin (H4B). Isolation of active enzyme required the coexpression of calmodulin. Recombinant holoNOS displayed similar spectral characteristics and activity as the enzyme isolated from murine macrophages. H4B-free NOS exhibited a Soret band at approximately 420 nm and, by analytical gel filtration, consisted of a mixture of monomers and dimers. H4B-free NOS catalyzed the oxidation of NG-hydroxy-L-arginine (NHA) with either hydrogen peroxide (H2O2) or NADPH and O2 as substrates. No product formation from arginine was observed under either condition. The amino acid products of NHA oxidation in both the H2O2 and NADPH/O2 reactions were determined to be citrulline and Ndelta-cyanoornithine (CN-orn). Nitrite and nitrate were also formed. Chemiluminescent analysis did not detect the formation of nitric oxide (*NO) in the NADPH/O2 reaction. The initial inorganic product of the NADPH/O2 reaction is proposed to be the nitroxyl anion (NO-) based on the formation of a ferrous nitrosyl complex using the heme domain of soluble guanylate cyclase as a trap, and the formation of a ferrous nitrosyl complex of H4B-free NOS during turnover of NHA and NADPH. NO- is unstable and, under the conditions of the reaction, is oxidized to nitrite and nitrate. At 25 degreesC, the H2O2-supported reaction had a specific activity of 120 +/- 14 nmol min-1 mg-1 and the NADPH-supported reaction had a specific activity of 31 +/- 6 nmol min-1 mg-1 with a KM,app for NHA of 129 +/- 9 microM. HoloNOS catalyzed the H2O2-supported reaction with a specific activity of 815 +/- 30 nmol min-1 mg-1 and the NADPH-dependent reaction to produce *NO and citrulline at 171 +/- 20 nmol min-1 mg-1 with a KM, app for NHA in the NADPH reaction of 36.9 +/- 0.3 microM.

Inhibition of murine macrophage nitric oxide synthase expression by a pivoxil prodrug of antiviral acyclic nucleotide analogue 9-(2-phosphonomethoxyethyl)adenine

The effect of the acyclic nucleotide analogue, 9-(2-phosphonomethoxyethyl)adenine (PMEA, Adefovir), and its (bis)pivaloyloxymethyl ester (bis-POM-PMEA, Adefovir Dipivoxil) on in vitro nitric oxide (NO) production by murine peritoneal macrophages was investigated. Bis-POM-PMEA inhibited in a concentrationdependent manner the formation of NO generated by interferon-γ and lipopolysaccharide, the 1C 50 being 15 μM. Suppressed transcription of mRNA for inducible NO synthase (EC 1.14.13.39) resulting in decreased synthesis of NO synthase protein was found. Parent compound PMEA was virtually ineffective.

Hyaluronan Fragments Induce Nitric-oxide Synthase in Murine Macrophages through a Nuclear Factor κB-dependent Mechanism

Activated macrophages play a critical role in controlling chronic tissue inflammation through the release of a variety of mediators including cytokines, chemokines, growth factors, active lipids, reactive oxygen, and nitrogen species. The mechanisms that regulate macrophage activation in chronic inflammation are poorly understood. A hallmark of chronic inflammation is the turnover of extracellular matrix components, and recent work has suggested that interactions with the extracellular matrix can exert important influences on macrophage effector functions. We have examined the effect of low molecular weight fragments of the extracellular matrix glycosaminoglycan hyaluronan (HA) on the induction of nitric-oxide synthase (iNOS) in macrophages. We found that HA fragments induce iNOS mRNA, protein and activity alone, and markedly synergize with interferon-gamma to induce iNOS gene expression in murine macrophages. In addition, we found that resident tissue alveolar macrophages respond minimally, but inflammatory alveolar macrophages exhibit a marked induction in iNOS expression in response to HA fragments. Finally, we demonstrate that the mechanism of HA fragment-induced expression of iNOS requires activation of the transcriptional regulator nuclear factor kappaB. These data support the hypothesis that HA may be an important regulator of macrophage activation at sites of chronic tissue inflammation.

Inhibition of hemoglobin expression by heterologous production of nitric oxide synthase in the K562 erythroleukemic cell line

Recent studies have indicated that nitric oxide may affect iron metabolism through disruption of the iron-sulfur complex of iron regulatory protein-1, a translational regulator. In the present study, we report that heterologous expression of murine macrophage nitric oxide synthase (NOS-2) in the human erythroleukemic K562 cell line results in constitutive production of nitric oxide associated with inhibition of hemoglobin expression. K562 cells were transfected with an episomally-maintained, hygromycin-selectable expression vector bearing the coding region of NOS-2. Constitutive NOS expression was detected by Western blotting of cell lysates and by the accumulation of nitrite in the culture media. Although NOS-transfected cells grew more slowly than control cells, they were able to maintain constitutive expression of NOS and production of nitric oxide for more than 1 month following transfection. The hemoglobin content of NOS-transfected K562 cells was less than one-fifth that of control cells, but increased markedly if NOS inhibitor was included in the culture media. The nitric oxide-mediated inhibition of hemoglobin expression was reversed by supplementing the culture media with 20 mumol/L hemin or 0.5 mmol/L 5- amino-levulinate, indicating that nitric oxide did not directly inhibit hemoglobin synthesis, but likely acted on a step in heme synthesis. mRNA levels for globin and erythroid aminolevulinic acid synthase (eALAS) were the same in both NOS-transfected and control cells. Our observations indicate that hemoglobin expression is inhibited by nitric oxide in NOS-transfected K562 cells by posttranscriptional repression of eALAS, the first enzyme of the heme biosynthetic pathway. The most likely mechanism is a nitric oxide-mediated translational repression of eALAS, as was recently demonstrated for ferritin synthesis. These observations further illustrate the potential for endogenously produced nitric oxide to regulate cellular posttranscriptional events. In particular, our observations may be relevant to the role of nitric oxide in anemia and lowered blood hemoglobin concentrations that are associated with chronic infections, such as tuberculosis or parasitic disease.

Inhibition of Hemoglobin Expression by Heterologous Production of Nitric Oxide Synthase in the K562 Erythroleukemic Cell Line

Recent studies have indicated that nitric oxide may affect iron metabolism through disruption of the iron-sulfur complex of iron regulatory protein-1, a translational regulator. In the present study, we report that heterologous expression of murine macrophage nitric oxide synthase (NOS-2) in the human erythroleukemic K562 cell line results in constitutive production of nitric oxide associated with inhibition of hemoglobin expression. K562 cells were transfected with an episomally-maintained, hygromycin-selectable expression vector bearing the coding region of NOS-2. Constitutive NOS expression was detected by Western blotting of cell lysates and by the accumulation of nitrite in the culture media. Although NOS-transfected cells grew more slowly than control cells, they were able to maintain constitutive expression of NOS and production of nitric oxide for more than 1 month following transfection. The hemoglobin content of NOS-transfected K562 cells was less than one-fifth that of control cells, but increased markedly if NOS inhibitor was included in the culture media. The nitric oxide-mediated inhibition of hemoglobin expression was reversed by supplementing the culture media with 20 mumol/L hemin or 0.5 mmol/L 5-amino-levulinate, indicating that nitric oxide did not directly inhibit hemoglobin synthesis, but likely acted on a step in heme synthesis. mRNA levels for globin and erythroid aminolevulinic acid synthase (eALAS) were the same in both NOS-transfected and control cells. Our observations indicate that hemoglobin expression is inhibited by nitric oxide in NOS-transfected K562 cells by posttranscriptional repression of eALAS, the first enzyme of the heme biosynthetic pathway. The most likely mechanism is a nitric oxide-mediated translational repression of eALAS, as was recently demonstrated for ferritin synthesis. These observations further illustrate the potential for endogenously produced nitric oxide to regulate cellular posttranscriptional events. In particular, our observations may be relevant to the role of nitric oxide in anemia and lowered blood hemoglobin concentrations that are associated with chronic infections, such as tuberculosis or parasitic disease.

Interaction of calmodulin with the inducible murine macrophage nitric oxide synthase.

The inducible isoform of nitric oxide synthase (iNOS) isolated from murine macrophages copurifies with calmodulin (CaM) as a tightly bound subunit. The exact function of this tightly bound CaM, however, has not been elucidated. In order to probe the function of this unusually strong interaction between iNOS and CaM, a 30 amino acid peptide derived from the putative CaM binding site of iNOS was synthesized. Cross-linking and autoradiographic analyses demonstrated that the peptide and CaM form a 1:1 complex as well as several higher molecular weight complexes. When assayed in the presence of a 12-fold excess of peptide to iNOS, over 90% of the enzymatic activity was inhibited. This inhibition could be prevented with the addition of exogenous bovine CaM to the assay mixture, in a concentration-dependent manner. Native PAGE and Western blot analysis of iNOS treated with peptide revealed the formation of a peptide--CaM complex with CaM derived from iNOS. Moreover, EGTA (5 mM) caused a 30% maximal inhibition of activity that was reversed by the addition of exogenous Ca2+ in a concentration-dependent fashion, suggesting a role for Ca2+ in this interaction. EGTA also changed the native PAGE mobility of iNOS and increased the intensity of a band which comigrates with CaM. These results demonstrate that the binding interaction between CaM and iNOS is tight but reversible, requires Ca2+, and is atypical from other known CaM--enzyme interactions.

Hydrogen peroxide-supported oxidation of NG-hydroxy-L-arginine by nitric oxide synthase.

The ability of murine macrophage nitric oxide synthase (NOS) to utilize peroxides in place of O2 and NADPH was investigated using hydrogen peroxide (H2O2), tert-butylhydroperoxide, and cumene hydroperoxide with both L-arginine and NG-hydroxy-L-arginine (L-NHA) as substrates. Of the three peroxides examined, only H2O2 was able to support product formation using L-NHA as a substrate. No product formation was observed from L-arginine with any peroxide tested. Therefore, the L-NHA/H2O2 reaction was examined in greater detail. The products of the reaction were citrulline and nitrite/nitrate (NO2-/NO3-) with a stoichiometry of approximately 0.75:1 (citrulline to NO2-/NO3-). Product formation was greater in the presence of oxygen. Both the Km and Vmax of the reaction, determined under aerobic conditions, were affected by (6R)-tetrahydro-L-biopterin (H4B). Chemiluminescence experiments failed to detect nitric oxide (.NO) as a reaction product. However, spectral spectral experiments with L-NHA and H2O2 under anaerobic conditions demonstrated the appearance of a ferrous heme-.NO complex with a Soret peak at 440 nm and a broad single alpha/beta peak at 578 nm, which is believed to arise from single electron transfer of a ferric-NO- (nitroxyl) complex. Preliminary experiments detected nitrous oxide (N2O) formation by gas chromatography under anaerobic conditions. Stable isotope labeling experiments with [18O]H2O2 conclusively established incorporation of label exclusively into the ureido position of citrulline. Based on these results, a mechanism of oxidation of L-NHA by H2O2 is proposed.

Interferon-γ-Inducible Murine Macrophage Nitric-Oxide Synthase: Studies on the Mechanism of Inhibition by Imidazole Agents

Citrulline formation by the interferon-γ/lipopolysaccharide-inducible murine macrophage nitric oxide synthase is inhibited reversibly by imidazole, 1-phenylimidazole, 4-phenylimidazole, and 2-phenylimidazole with IC50 values of 40 μM, 6 μM, 225 μM and >1 mM, respectively. 1-Phenylimidazole inhibited the maximal velocity of citrulline formation but did not alter the concentration of arginine providing half-maximal activity. 1-Phenylimidazole inhibited citrulline formation by the murine macrophage nitric oxide synthase competitively versus (6R)-5,6,7,8-tetrahydro-L-biopterin (THB) with a Ki value of 0.7 μM, but inhibited citrulline formation by Ca2+-calmodulin-dependent nitric oxide synthase from GH3 pituitary cells noncompetitively versus THB with a Ki value of 40 μM. Imidazole inhibited citrulline formation by the murine macrophage nitric oxide synthase noncompetitively versus THB with a Ki value of 48 μM. Neither imidazole nor 1-phenylimidazoIe inhibited the cytochrome c reductase activity of murine macrophage nitric oxide synthase at concentrations 100-fold higher than their IC50 values for inhibiting citrulline formation. The antifungal imidazoles miconazole, ketoconazole, and clotrimazole did not inhibit either citrulline formation or cytochrome c reduction by murine macrophage nitric oxide synthase at concentration as high as 200 μM. Ca2+-calmodulin-dependent nitric oxide synthase from GH3 pituitary cells exhibited a Kact for THB of 80 nM, while the inducible murine macrophage nitric oxide synthase exhibited a Kact of 8 μM.

Irreversible inactivation of macrophage and brain nitric oxide synthase by L-NG-methylarginine requires NADPH-dependent hydroxylation.

L-NG-Methylarginine (NMA) is an established mechanism-based inactivator of murine macrophage nitric oxide synthase (mNOS). In this report, NMA is shown to irreversibly inhibit both mNOS (k(inact) = 0.08 min-1) and the recombinant constitutive brain NOS (bNOS). For both NOS isoforms, metabolism of NMA parallels that of the natural substrate L-arginine (ARG), in that it undergoes a regiospecific, NADPH-dependent hydroxylation to form L-NG-hydroxy-NG-methylarginine (NOHNMA). This intermediate then undergoes further NADPH-dependent oxidation to form L-citrulline (CIT). Authentic NOHNMA, synthesized from L-ornithine, irreversibly inhibited both mNOS (k(inact) = 0.10 min-1) and bNOS in an NADPH-dependent reaction. The conversion of either NMA or NOHNMA to CIT correlated with irreversible enzyme inactivation. Thus, the data suggest that enzyme inhibition occurs as a consequence of oxidative metabolism of the intermediate, NOHNMA. A unified mechanism is proposed that accounts for NO biosynthesis from ARG, for the inactivation of NOS by NMA and for the intermediacy of hydroxylated ARG or NMA derivatives in these processes.

Cloned, expressed rat cerebellar nitric oxide synthase contains stoichiometric amounts of heme, which binds carbon monoxide.

The endogenous formation of nitric oxide (NO) has become an area of intense interest as evidence for its biological functions has been obtained in three distinct tissues: circulating macrophages, in which it exerts cytotoxic effects; blood vessels, in which it has been identified as endothelium-derived relaxing factor; and neuronal cells, in which it functions as a neurotransmitter. The formation of NO in brain extracts has been shown to be catalyzed by an enzyme, termed NO synthase, which generates the NO responsible for stimulation of cGMP formation, the highest levels of which occur in the cerebellum. NO synthase catalyzes the formation of citrulline from arginine with the coincident production of NO and has been shown to be a flavoprotein, containing 1 mol each of FAD and FMN, tetrahydrobiopterin, and iron. It is also reported to contain an alpha-helical, calmodulin-binding consensus sequence consistent with its stimulation by calmodulin in the presence of Ca2+. The formation of NO requires incorporation of one of the atoms of molecular oxygen into one of the guanidinium nitrogen atoms of arginine with the coincident formation of citrulline. This communication reports that rat cerebellar NO synthase, cloned and stably expressed in human kidney 293 cells, contains heme in amounts stoichiometric with the flavins FAD and FMN as evidenced by the appearance of a pyridine hemochrome and a reduced CO difference spectrum with an absorbance maximum at approximately 445 nm. The finding of a CO-binding heme moiety explains the presence of iron in the enzyme and suggests a role for prosthetic heme as an oxygenase reaction center. This report also presents evidence for incorporation of delta-[14C]aminolevulinate specifically into immunoprecipitable NO synthase in stably transfected human kidney 293 cells but not in nontransfected cells. Simultaneously, K. A. White and M. A. Marletta [(1992) Biochemistry 31, 6627-6631] have demonstrated a CO-binding heme prosthetic group in purified murine macrophage NO synthase and have suggested the identity of these reaction centers in both the constitutive (cerebellar) and inducible (macrophage) forms of NO synthase.

Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein.

The synthesis of nitric oxide (.NO) from L-arginine has been demonstrated in a number of cell types and functions either as a cell signaling agent or as a key component of the cell-mediated immune response. Both constitutive and inducible activities have been described. Herein we report the purification of inducible .NO synthase (EC 1.14.23) from activated murine macrophages using a two-column procedure. Crude 100,000 x g supernatant was passed through a 2'-5'-ADP-Sepharose 4B affinity column followed by a DEAE-Bio-Gel A anion exchange column. The .NO synthase ran as a band of Mr = 130,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration experiments using a Superose 6 HR 10/30 column estimated the native molecular weight to be 260 +/- 30 kDa, indicating that the native enzyme exists as a dimer. Activity was dependent upon L-arginine (Km = 16 +/- 1 microM at 37 degrees C and pH 7.5) and NADPH. Both (6R)-tetrahydro-L-biopterin and FAD enhanced activity, whereas Mg2+ and FMN had no effect on activity. Fluorescence studies demonstrated the presence of one bound FAD and one bound FMN per subunit.