Nitric Oxide In Murine Macrophages Research Articles

Carbon monoxide-releasing molecule-401, a water-soluble manganese-based metal carbonyl, suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide in murine macrophages

Context Many reports in the literature have suggested the therapeutic value of carbon monoxide-releasing molecules (CORMs) against various diseases. However, to date, little is known about their possible influence on periodontal disease. Objective This study was performed to investigate the influence of CORM-401 on the generation of nitric oxide (NO) in murine macrophage cells activated with lipopolysaccharide (LPS) derived from Prevotella intermedia, a pathogen associated with periodontal disease. Materials and methods LPS was isolated by the hot phenol-water method. Culture supernatants were analyzed for NO. Real-time PCR and immunoblotting were conducted to quantify mRNA and protein expression, respectively. NF-κB-dependent SEAP levels were estimated by reporter assay. DNA-binding of NF-κB was also analyzed. Results CORM-401 caused an apparent suppression of NO production through inhibition of iNOS at both the mRNA and protein levels in RAW264.7 cells stimulated with P. intermedia LPS. CORM-401 upregulated the expression of both the HO-1 gene and its protein in LPS-activated cells, and treatment with the HO-1 inhibitor significantly reversed the attenuating influence of CORM-401 against LPS-induced generation of NO. CORM-401 caused an apparent attenuation of NF-κB-dependent SEAP release induced by LPS. IκB-α degradation and nuclear translocation of NF-κB p50 subunit induced by LPS were significantly reduced by CORM-401. Additionally, CORM-401 significantly attenuated DNA-binding of p65 and p50 induced by LPS. CORM-401 attenuated NO generation induced by P. intermedia LPS independently of PPAR-γ, JNK, p38 and STAT1/3. Conclusion The modulation of host inflammatory response by CORM-401 might be of help in the therapy of periodontal disease.

Effect of quercetin on the production of nitric oxide in murine macrophages stimulated with lipopolysaccharide fromPrevotella intermedia

PurposeNitric oxide (NO) is a short-lived bioactive molecule that is known to play an important role in the pathogenesis of periodontal disease. In the current study, we investigated the effect of the flavonoid quercetin on the production of NO in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen related to inflammatory periodontal disease, and tried to elucidate the underlying mechanisms of action.MethodsLPS was isolated from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The concentration of NO in cell culture supernatants was determined by measuring the accumulation of nitrite. Inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) protein expression, phosphorylation of c-Jun N-terminal kinase (JNK) and p38, inhibitory κB (IκB)-α degradation, and signal transducer and activator of transcription 1 (STAT1) phosphorylation were analyzed via immunoblotting.ResultsQuercetin significantly attenuated iNOS-derived NO production in RAW246.7 cells activated by P. intermedia LPS. In addition, quercetin induced HO-1 protein expression in cells activated with P. intermedia LPS. Tin protoporphyrin IX (SnPP), a competitive inhibitor of HO-1, abolished the inhibitory effect of quercetin on LPS-induced NO production. Quercetin did not affect the phosphorylation of JNK and p38 induced by P. intermedia LPS. The degradation of IκB-α induced by P. intermedia LPS was inhibited when the cells were treated with quercetin. Quercetin also inhibited LPS-induced STAT1 signaling.ConclusionsQuercetin significantly inhibits iNOS-derived NO production in murine macrophages activated by P. intermedia LPS via anti-inflammatory HO-1 induction and inhibition of the nuclear factor-κB and STAT1 signaling pathways. Our study suggests that quercetin may contribute to the modulation of host-destructive responses mediated by NO and appears to have potential as a novel therapeutic agent for treating inflammatory periodontal disease.

Low molecular weight hyaluronic acid effects on murine macrophage nitric oxide production

Hyaluronic acid (HA) is increasingly used for a number of medical device applications. Since the chemical structure of HA is identical no matter its bacterial or animal origin, it should be the ideal biomaterial. However, short term transient inflammatory reactions are common, while rare long-term adverse events may correlate with subclinical chronic inflammation. Concern has been raised that low molecular weight components or degradation fragments from implanted HA may directly stimulate inflammatory reactions. This study examined a panel of HA molecular weights from the unitary disaccharide up to 1.7 x 10(6) Dalton lengths, in which endotoxin was assayed at a very low level (less than 0.03 EU/mg). The murine cell line RAW 264.7, rat splenocytes, and rat adherent differentiated primary macrophages were assayed for nitric oxide production under a variety of inflammatory conditions plus or minus HA. Under the highest inflammatory states, nitric oxide production was mildly suppressed by HMW-HA while slightly augmented by LMW-HA at mg/mL concentrations. However, at micromolar concentrations fragments below 5000 Daltons, thought to have drug-like qualities, were without effect. These data support the hypothesis that if endotoxin is reduced to an extremely low level, LMW-HA may not directly provoke normal tissue macrophage-mediated inflammatory reactions.

Dual β-adrenergic modulation in the immune system: Stimulus-dependent effect of isoproterenol on MAPK activation and inflammatory mediator production in macrophages

This is the first study to demonstrate that the interaction between β-adrenoceptor activation, and the production of inflammatory mediators can be modulated in opposite ways by two inflammatory stimuli, namely, protein kinase C (PKC)-activating phorbol myristyl acetate (PMA) and lipopolysaccharide (LPS). We provided evidence that isoproterenol treatment, when combined with phorbol ester increased the production of tumor necrosis factor-alpha, interleukin-12, and nitric oxide in murine macrophages, as well as in human monocytes and differentiated PLB-985 cells, while in agreement with earlier findings, it decreased inflammatory mediator production in combination with LPS stimulation. The contrasting effect on inflammatory mediator production, shown for the PMA and LPS activated cells was accompanied by parallel changes in activation of ERK1/2 and p38 MAPKs. Thus, isoproterenol significantly increased MAPK activation (phosphorylation) in PMA-treated cells and, conversely, it decreased the activation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 in LPS-stimulated cells. The opposing effects of isoproterenol on LPS-induced versus PMA-induced mediator production and the concurrent changes in MAPK activation highlight the role of this kinase pathway in macrophage activation and provide new insights regarding the flexible ways through which β-adrenoceptor stimulation can modulate the inflammatory response in macrophages. Our results challenge the dogma that β-adrenoceptor signaling is only immunosuppressive, and offer potential opportunities for new therapeutic approaches in the treatment of inflammatory and autoimmune diseases.

Leishmania lipophosphoglycan activates the transcription factor activating protein 1 in J774A.1 macrophages through the extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase

Leishmania donovani is an obligatory intracellular pathogen that resides and multiplies in the phagolysosomes of macrophages. The outcome of this infection depends on the balance between the host ability to activate macrophage killing and the parasite ability to suppress or evade this host immune response. Lipophosphoglycan (LPG) glycoconjugate, the surface molecule of the protozoan parasite is a virulence determinant and a major parasite molecule involved in this process. In this study, we examined the ability of Leishmania and its surface molecule, lipophosphoglycan to activate activating protein 1 (AP-1) through the mitogen-activated protein kinase (MAPK) cascade. We report here that the Leishmania surface molecule, lipophosphoglycan stimulates the simultaneous activation of all three classes of MAP kinases, extracellular signal-related kinases (ERKs), the c-jun amino-terminal kinase (JNK) and the p38 MAP kinase with differential kinetics in J774A.1 macrophage cell line. Furthermore, both L. donovani and its surface molecule lipophosphoglycan resulted in a dose- and time-dependent induction of AP-1 DNA-binding activity. We have also shown a dose-dependent increase of AP-1 binding activity in both low and high virulent strains of parasite. The use of inhibitors selective for ERK (PD98059) and p38 (SB203580) pathway showed that pre-incubation of cells with either SB203580 or PD98059 affected the binding activity of AP-1 suggesting that both p38 and ERK MAP kinase activation appear to be necessary for AP-1 activation by LPG. Lipophosphoglycan induced IL-12 production and generation of nitric oxide in murine macrophages. These results demonstrate that L. donovani LPG activates pro-inflammatory, endotoxin-like response pathway in J774A.1 macrophages and the interaction may play a pivotal role in the elimination of the parasite.

Effect of melatonin on the generation of nitric oxide in murine macrophages

In the present investigation, the effects of melatonin on the generation of nitric oxide in stimulated murine macrophages were studied by electron paramagnetic resonance techniques, and the underlying mechanisms were discussed. Upon stimulation with lipopolysaccharide/interferon-γ, macrophages generated a high concentration of nitric oxide. Melatonin pretreatment significantly inhibited the generation of nitric oxide. Western blot and reverse transcription polymerase chain reaction analysis showed that melatonin decreased the expression of inducible nitric oxide synthase at either the protein or mRNA level. Further investigation showed that melatonin significantly attenuated the nitration of cytoplasmic IκB-α, inhibited the degradation of IκB-α, and blocked the translocation of p65/RelA into the nuclei. The results suggest that melatonin inhibits the expression of inducible nitric oxide synthase by modulating the nuclear factor κB pathway.

Different effects of ferulic acid and tetramethylpyrazine on the production of superoxide anion and nitric oxide in murine macrophages

The effects of ferulic acid and tetramethylpyrazine, two active ingredients of the Chinese herbal medicineLigusticum wallichii Franchat, on the generation of superoxide anion and nitric oxide in macrophages were studied. Ferulic acid, but not tetramethylpyrazine, scavenges superoxide anion dose-dependently. Tetramethylpyrazine inhibits the expression of the iNOS gene, and consequently decreases the formation of nitric oxide. However, ferulic acid shows no effect on iNOS expression and NO production. The results suggest that the protective effects of extracts ofLigusticum wallichi Franchat against ischemic injury might be due to the scavenging of superoxide anion and the regulation of NO production.

Inhibition of interleukin-12 p40 transcription and NF-kappaB activation by nitric oxide in murine macrophages and dendritic cells.

Nitric oxide (NO), an important effector molecule of the innate immune system, can also regulate adaptive immunity. In this study, the molecular effects of NO on the toll-like receptor signaling pathway were determined using interleukin-12 (IL-12) as an immunologically relevant target gene. The principal conclusion of these experiments is that NO inhibits IL-1 receptor-associated kinase (IRAK) activity and attenuates the molecular interaction between tumor necrosis factor receptor-associated factor-6 and IRAK. As a consequence, the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibits lipopolysaccharide (LPS)-induced IL-12 p40 mRNA expression, protein production, and promoter activity in murine macrophages, dendritic cells, and the murine macrophage cell line RAW 264.7. Splenocytes from inducible nitric-oxide synthase-deficient mice demonstrate markedly increased IL-12 p40 protein and mRNA expression compared with wild type splenocytes. The inhibitory action of NO on IL-12 p40 is independent of the cytokine IL-10. The effects of NO can be directly attributed to inhibition of NF-kappaB activation through IRAK-dependent pathways. Accordingly, SNAP strongly reduces LPS-induced NF-kappaB DNA binding to the p40 promoter and inhibits LPS-induced IkappaB phosphorylation. Similarly, NO attenuates IL-1beta-induced NF-kappaB activation. These experiments provide another example of how an innate immune molecule may have a profound effect on adaptive immunity.

Inhibition of murine macrophage nitric oxide production by synthetic oligonucleotides

Synthetic 30-mer phosphorothioate (Ps) oligonucleotides (ODN) comprised of single bases (SdA30, SdC30, SdG30, and SdT30) were assessed for their effects on nitric oxide (NO) production by murine bone marrow macrophages (BMMC) and macrophage cell lines J774 and RAW264.7. Pretreatment of these cells with any of the four Ps ODN inhibited NO production induced by CpG ODN, E. coli DNA (EC DNA), or LPS. This inhibition was time- and dose-dependent and was observed even if the Ps ODN were added as long as 12 h after stimulation. As in the case of stimulatory ODN, inhibition was dependent on backbone structure and length. Thus, all four 30-mer, single-base Ps ODN were inhibitory, and only dG30 among phosphodiester ODN was inhibitory. Together, these observations indicate that Ps ODN can inhibit macrophage production of inflammatory mediators, suggesting a role of these compounds as immunomodulatory agents.

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.

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.

Evidence for murine macrophage nitric oxide synthesis in the absence of exogenous L-arginine.

Evidence for murine macrophage nitric oxide synthesis in the absence of exogenous L-arginine.

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.

Activation of inducible nitric oxide synthase gene in murine macrophages requires protein phosphatases 1 and 2A activities.

The purpose of these studies was to identify phosphatase activities required for the production of nitric oxide in murine macrophages exposed to lipopolysaccharide (LPS), synthetic lipopeptide (LPP), and mouse interferon-gamma (IFN-gamma). The in vitro treatment of macrophages with IFN-gamma and LPS or IFN-gamma and LPP resulted in production of NO, which was inhibited by addition of the specific phosphatase 1 and 2A (PP1/2A) inhibitors okadaic acid (OA), calyculin A, and cantharidin (but not the nonactive analogues okadaic acid tetraacetate and 1,4-dimethylendothall). OA suppressed the accumulation of steady-state inducible NO synthase (iNOS) mRNA and iNOS protein (without alteration of their stability). The cytosol and nuclei of control macrophages contained large amounts of PP1/2A activities that were inhibited by OA in a dose-dependent manner. Taken together, these data indicate that PP1/2A activities are involved in the regulation of iNOS gene expression in murine macrophages.

Agonistic and antagonistic activities of bacterially derived Rhodobacter sphaeroides lipid A: comparison with activities of synthetic material of the proposed structure and analogs

Lipid A from the photosynthetic bacterium Rhodobacter sphaeroides (RSLA) has been previously shown to antagonize many of the effects of endotoxins from more pathogenic gram-negative bacteria. We have reported on the synthesis of the proposed structure of RSLA and determined that bacterially derived RSLA is not identical to its proposed structure (W.J. Christ, P. D. McGuinness, O. Asano, Y. Wang, M. A. Mullarkey, M. Perez, L. D. Hawkins, T. A. Blythe, G. R. Dubuc, and A. L. Robidoux, J. Am. Chem. Soc. 116:3637-3638, 1994). Here we report results of analyzing the antagonistic and agonistic activities of bacterially derived RSLA in comparison with the activities of chemically synthesized material of the proposed structure of RSLA and analogs. Results indicated that all compounds were approximately equally potent at inhibiting endotoxin-induced release of tumor necrosis factor alpha from human monocytes and human whole blood as well as endotoxin-induced generation of nitric oxide in murine macrophages. In addition, all compounds were of equivalent potencies at inhibiting the binding of 125I-labelled lipopolysaccharide derivatized with 2-(p-azido-salicylamido) ethyl-1-3'-dithiopropionate to murine macrophages. Higher concentrations of bacterially derived RSLA (10 to 100 microM) were agonistic in human and murine assays. In gamma interferon-treated murine macrophages, agonism was exhibited at concentrations as low as 100 nM. In contrast, all synthetic materials were either dramatically less agonistic or devoid of agonistic activity when tested at concentrations as high as 100 microM. It is possible either that bacterially derived RSLA contains a small amount of a highly agonistic impurity or that the agonistic activity of RSLA is intrinsic to its molecular structure. In either case, these biological results support our previous report concluding that biologically derived RSLA is not identical to synthetic material of its proposed structure.

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.

Modulation of Murine Macrophage Nitric Oxide Synthesis by Liposomal Phospholipids: Correlation with Liposome Immune Adjuvant Activity

The influence of alum and liposomal phospholipids on interferon-gamma-(IFN-gamma), IFN-gamma/N-acetylmuramyl-L-alanyl-D-isoglutamine- (MDP) or IFN-gamma/tumor necrosis factor-alpha- (IFN-gamma/TNF-alpha) induced macrophage nitric oxide (NO) synthesis has been investigated. IFN-gamma induced NO synthesis in a dose-dependent manner. TNF-alpha and MDP did not induce NO synthesis, but interacted synergistically with sub-optimal doses of IFN-gamma. Alum strongly inhibited IFN-gamma-induced NO synthesis (ID50 25 microgram/ml). Liposomes composed of dipalmitoylphosphatidylcholine (DPPC) had no effect on IFN-gamma-induced NO synthesis. IFN-gamma-induced NO synthesis was stimulated by DPPC/dimyristoylphosphatidylglycerol (DMPG) liposomes (9:1 mol ratio, ED50 45 nmol phospholipid/ml), and inhibited by DPPC/dipalmitoylphosphatidylethanolamine (DPPE) liposomes (9:1 mol ratio, ID50 > 500 nmol phospholipid/ml), and DPPC/phosphatidylserine (PS) liposomes (7:3 mol ratio, ID50 150 nmol phospholipid/ml). Alum, DPPC/PE and DPPC/PS liposomes also inhibited IFN-gamma/MDP- and IFN-gamma/TNF-alpha-induced NO synthesis. Neither alum or the liposome preparations had significant toxicity towards macrophages in vitro at concentrations that induced maximal inhibition or stimulation of IFN-gamma-induced NO synthesis. Immunization of mice with alum-adsorbed and liposome-incorporated bovine serum albumin (BSA) demonstrated that enhancement or reduction of both IgG antibody and the proportion of IgG2a/IgG2b was correlated with stimulation or inhibition of IFN-gamma-induced NO synthesis.