IFN-gamma-induced TNF-alpha is a prerequisite for in vitro production of nitric oxide generated in murine peritoneal macrophages by IFN-gamma

Dopamine is formed form L-tyrosine by tyrosine hydroxylase and aromatic L-amino acid decarboxylase. In addition to this pathway, however, the formation of catecholamines, including dopamine, from trace amines such as tyramine by hepatic microsomes has been demonstrated. In this study, we investigated the formation of dopamine from trace amines, using human hepatic microsomes and human cytochrome P450 (CYP) isoforms expressed in yeast. Among the 11 isoforms of human CYP expressed in yeast, CYP2D6 was the only isoform exhibiting strong ability to convert p-tyramine and m-tyramine to dopamine. In studies with human hepatic microsomes, the hydroxylation of tyramine to dopamine was inhibited by bufuralol, a typical substrate for CYP2D isoforms, and anti-CYP2D1 antiserum. This is the first report showing that CYP2D is capable of converting tyramine to dopamine. The Km values of CYP2D6, expressed in yeast, for p-tyramine and m-tyramine were 190.1 +/- 19.5 microM and 58.2 +/- 13.8 microM, respectively. Tyramine is an endogenous compound which exists in the brain as a trace amine but is also an exogenous compound which is found in foods such as cheese and wine. Our results suggest that dopamine is formed from endogenous and/or exogenous tyramine by this CYP2D isoform.     

Aflatoxin B1-induced suppression of nitric oxide production in murine peritoneal macrophages

Aflatoxin B1 (AFB1), a potent hepatocarcinogen, is known to impair specific and non-specific immune responses. AFB1 mainly decreases lymphocyte functions and may also affect macrophages assisting lymphocyte functions. Macrophages play an important role in a host defense against tumors and bacteria. Furthermore, some macrophage products, including nitric oxide (NO), may be involved in cytotoxicity. The effect of aflatoxin B1 (AFB1) was investigated on NO production from murine peritoneal macrophages. Macrophages were pretreated with AFB1 for 24 h and then stimulated with lipopolysaccharide (LPS) for 24 h. AFB1 at 10 or 50 microM reduced the production of NO. Compared to vehicle control, there was a greater reduction of NO production with increased AFB1 pretreatment and LPS stimulation. AFB1 at 10 or 50 microM decreased inducible nitric oxide synthase (iNOS) activity about 24% and 28%, respectively, after stimulation with 1 microg/ml LPS and about 12% and 24%, respectively, after stimulation with 10 microg/ml LPS. AFB1 pretreatment also decreased the synthesis of iNOS protein and the mRNA of macrophages. Taken together, these results suggest that AFB1 pretreatment reduces NO production from murine peritoneal macrophages stimulated by LPS, which is mediated by the reduction of iNOS activity, mRNA, and protein.     

The triterpenoid quinonemethide pristimerin inhibits induction of inducible nitric oxide synthase in murine macrophages

Inducible nitric oxide synthase dependent production of nitric oxide (NO) plays an important role in inflammation. We investigated whether pristimerin ((20alpha)-3-hydroxy-2-oxo-24-nor-friedela-1(10),3,5,7-te traen-carboxylic acid-(29)-methylester), an antitumoral, antimicrobial as well as anti-inflammatory plant compound, has an effect on the inducible NO synthase system in lipopolysaccharide-activated RAW 264.7 macrophages. Pristimerin dose dependently (IC50: 0.2-0.3 microM) reduces nitrite accumulation, a parameter for NO synthesis, in supernatants of lipopolysaccharide-stimulated (1 microg/ml, 20 h) macrophages. This effect correlates with a reduced inducible NO synthase enzyme activity measured by conversion of [3H]L-arginine to [3H]L-citrulline and significantly lower levels of enzyme protein (Western blotting) in homogenates of cells cotreated with lipopolysaccharide and pristimerin (12 h). Northern blot analysis and polymerase chain reaction (PCR) showed decreased inducible NO synthase mRNA levels in activated macrophages exposed to pristimerin (4 h). Electrophoretic mobility shift assay (EMSA) demonstrated a markedly reduced binding activity of nuclear factor-kappa B (NFkappaB) in nuclear extracts of pristimerin-treated cells. These results suggest that pristimerin inhibits the induction of inducible NO synthase by a mechanism which involves inhibition of NFkappaB activation. This feature of pristimerin is likely to contribute to its anti-inflammatory activity.     

An improved experimental model for the study of in vitro release of nitric oxide by murine peritoneal macrophages

This exposition analyses and contextualizes the complex problem of structural inequality in South African health care. Socio-economic conditions, racial divisions and geographical location are isolated as the main determinants of inequality in the provision, allocation and distribution of health care; the prevailing inequalities are attributed to a wide range of underlying causes, including the absence of a central, binding health policy, the prominent role of apartheid and white domination, the free market and the medical profession, as well as the unique sociocultural set-up of the country. The urgent need for deliberate strategies to equalize the prevailing disparities and discrepancies is posed.     

Pneumococci stimulate the production of the inducible nitric oxide synthase and nitric oxide by murine macrophages

The role of nitric oxide (NO) in the pathophysiology of gram-positive sepsis is uncertain. In inflammatory conditions, high-output NO production is catalyzed by the enzyme inducible nitric oxide synthase (iNOS). The ability of 2 strains of pneumococci, pneumococcal cell wall preparations, and purified pneumococcal capsule (Pnu-Imune 23) to trigger the production of iNOS protein and NO in RAW 264.7 murine macrophages was tested. Live pneumococci, oxacillin-killed pneumococci, and pneumococcal cell wall preparations stimulated the production of iNOS and NO by RAW 264.7 cells in the presence, but not the absence, of low concentrations of recombinant murine interferon-gamma. In contrast, purified pneumococcal capsule induced little or no iNOS or NO production by these cells. Thus, pneumococci stimulate high-output NO production by murine macrophages. The potential role of NO in the pathogenesis of pneumococcal sepsis deserves further study.     

The role of a phosphatidylcholine-specific phospholipase C in the production of diacylglycerol for nitric oxide synthesis in macrophages activated by IFN-gamma and LPS

Murine macrophages activated by interferon (IFN)-gamma and bacterial lipopolysaccharide (LPS) produce large amounts of nitric oxide (NO), which is a critical mediator for a variety of biological functions. The expression of this inducible NO synthase (iNOS) involves a protein kinase C (PKC)-dependent pathway, but the mechanism for the PKC activation in this system is unclear. Through analysis of diacylglycerol (DAG) synthesis and choline metabolism in activated macrophages, direct evidence is provided that NO synthesis involves the activation of an unusual phosphatidylcholine-specific phospholipase C (PC-PLC) and not a phosphatidylinositol-specific phospholipase C (PI-PLC) or phospholipase D (PLD).     

Synergistic induction of DNA strand breakage by cigarette tar and nitric oxide

Cigarette smoking is a major cause of human cancer at a variety of sites, although its carcinogenic mechanisms remains unestablished. Cigarette smoke can be divided into two phases, gas phase and particulate matter (tar). Both phases contain high concentrations of oxidants and free radicals, especially nitric oxide (NO) and nitrogen oxides in the gas phase and quinone/hydroquinone complex in the tar. We have found that incubation of pBR322 plasmid DNA with aqueous extracts of cigarette tar and a NO-releasing compound (diethylamine NONOate) caused synergistic induction of DNA single-strand breakage, whereas either cigarette tar alone or NO alone induced much less strand breakage. This synergistic effect of cigarette tar and NO on DNA strand breakage was prevented by high concentrations of superoxide dismutase, carboxy-PTIO (an NO-trapping agent) or N-acetylcysteine, whereas hydroxyl radical scavengers such as dimethylsulfoxide, ethanol and D-mannitol did not show inhibitory effects. Possible mechanisms for this synergistic effect mediated by cigarette tar and NO are proposed, including involvement of peroxynitrite, which is a strong oxidant and nitrating agent formed rapidly by the reaction between NO and O2.-. NO is present in the gas phase of smoke and may be formed by a constitutive or inducible NO synthase in the lung, whereas O2.- is generated by auto-oxidation of polyhydroxyaromatic compounds such as catechol and 1,4-hydroquinone present in cigarette tar. Thus, potent reactive species including peroxynitrite formed by the interaction between cigarette tar and NO may play an important role in smoking-related diseases including lung cancer.     

Horseradish peroxidase and glycosylated BSA induce nitric oxide production in murine macrophages

The in vitro activation of murine macrophages by horseradish peroxidase (HRP) induced nitric oxide production in a dose-dependent manner, and increased the induction of NO-synthase by LPS. Nitrite production after HRP stimulation was inhibited by NG-monomethyl-L-arginine (NMMA), a specific inhibitor of NO-synthase. Equivalent amounts of nitrite were obtained with native and heat-inactivated HRP. High concentrations of mannose inhibited nitric oxide production, while the HRP inhibitor 3-aminotyrosine did not. Glycosylated serum albumin derivatives also induced murine macrophage NOS, probably by an interaction between carbohydrates and their specific cell membrane receptors. The inability of HRP apoprotein to stimulate NO production, and the specific inhibition of HRP-mediated activation of macrophages by hemin suggests that the heme moiety of this enzyme is involved in NO-synthase induction.     

LPS from Actinobacillus actinomycetemcomitans and production of nitric oxide in murine macrophages J774

Nitric oxide (NO) plays a complex role in the modulation of the inflammatory response, having either a pro-inflammatory or a protective role. Actinobacillus actinomycetemcomitans is considered an important etiological agent in localized juvenile periodontitis. We have studied the effect of lipopolysaccharide (LPS) extracted from this periodontopathogenic bacterium on NO synthesis in an in vitro murine macrophage system. LPS from A. actinomycetemcomitans induced a significant production of NO even at concentrations as low as 1 ng/ml, whereas LPS from E. coli had to be added in concentrations of 100 ng/ml to obtain similar effects. Production of NO was blocked by NG-nitro-L-arginine methylester, and pre-treatment of LPS from A. actinomycetemcomitans with polymyxin B abolished the production of NO, while prostaglandin E2 enhanced the synthesis of NO.     

Differential nitric oxide and TNF-alpha production of murine Kupffer cell subfractions upon priming with IFN-gamma and TNF-alpha

The effects of a nitric oxide (NO) donor on microcirculation and contractile function of reperfused skeletal muscle were studied. Rat cremaster muscles underwent 5 hours of ischemia and 90 minutes of reperfusion and were divided into two groups systemically infused with S-nitroso-N-acetylcysteine (SNAC, 100 nmol/min) and phosphate-buffered saline (PBS), respectively. The results showed that the vessels in the SNAC group had more rapid and complete recovery than that in controls. A significant difference was found from 10 to 40 minutes and at 90 minutes in 10-20-microm arterioles, from 10 to 90 minutes in 20-40-microm arterioles, and at 10 and 90 minutes in 40-70-microm arteries. When compared to controls, SNAC-treated muscles showed larger fluorescein filling areas at 15, 30, 60, and 90 minutes and greater isometric tetanic contractile forces in response to stimulation frequencies of 40, 70, 100, and 120 Hz. The data indicate that supplementation of exogenous NO could effectively improve microcirculation and contractile function of skeletal muscle during early reperfusion.     

Effect of prolactin on nitric oxide and interleukin-1 production of murine peritoneal macrophages: role of Ca2+ and protein kinase C

The Psychiatric Hospital at the Municipal (General) Hospital in G?rlitz, Germany, was the only Department of Psychiatry in a non-University (general) hospital in the newly integrated German provinces who were originally part of the so-called "German Democratic Republic" before the re-unification of Germany, to participate in the German collective study on "Psychiatric and Psychosomatic Consultation and Liaison Service in German General Hospitals-A Multicentre Empirical Study to Assess and Evaluate Existing Structures and Services". This study is an independent part project that includes specific questions forming part of the European collective study on "Effectiveness of Mental Health Consultation and Liaison Service Delivery in the General Hospital".     

Phlebotomus papatasi saliva inhibits protein phosphatase activity and nitric oxide production by murine macrophages

Leishmania parasites, transmitted by phlebotomine sand flies, are obligate intracellular parasites of macrophages. The sand fly Phlebotomus papatasi is the vector of Leishmania major, a causative agent of cutaneous leishmaniasis in the Old World, and its saliva exacerbates parasite proliferation and lesion growth in experimental cutaneous leishmaniasis. Here we show that P. papatasi saliva contains a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A of murine macrophages. We further demonstrate that P. papatasi saliva down regulates expression of the inducible nitric oxide synthase gene and reduces nitric oxide production in murine macrophages. Partial biochemical characterization of the protein phosphatase and nitric oxide inhibitor indicated that it is a small, ethanol-soluble molecule resistant to boiling, proteolysis, and DNase and RNase treatments. We suggest that the P. papatasi salivary protein phosphatase inhibitor interferes with the ability of activated macrophages to transmit signals to the nucleus, thereby preventing up regulation of the induced nitric oxide synthase gene and inhibiting the production of nitric oxide. Since nitric oxide is toxic to intracellular parasites, the salivary protein phosphatase inhibitor may be the mechanism by which P. papatasi saliva exacerbates cutaneous leishmaniasis.     

The role of prostaglandin E2 and nitric oxide in cell death in J774 murine macrophages

We investigated the role of prostaglandin E2 (PGE2) and its interactions with nitric oxide (NO) on cell death and NO-mediated cytotoxicity in the murine macrophage cell line J774. Stimulation of the J774 cells with lipopolysaccharide together with interferon-gamma resulted in a dose-dependent cytotoxicity and production of PGE2 and NO, measured as nitrite. Our results showed a linear correlation between PGE2 release and cytotoxicity. The cyclooxygenase (COX) inhibitor indomethacin completely inhibited PGE2 biosynthesis, without affecting NO production or cell death. This supports previous reports suggesting that overproduction of endogenous PGE2 is mainly the consequence of cell death and does not cause it. In contrast, the NO synthase inhibitor N(omega)-monomethyl-L-arginine (L-NMMA) gave a significant, though incomplete suppression of NO release and cell death. This points to the presence of other cytotoxic factors besides NO. To evaluate the toxic effect solely due to NO, macrophages were exposed to the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP). Incubation with SNAP also resulted in a concentration-dependent cell injury and PGE2 production. When exogenously added, PGE2 protected against SNAP-mediated cytotoxicity and simultaneously increased PGE2 release into the medium, without inducing COX-2. The cytoprotection and the stimulation of PGE2 release were both reversed by indomethacin. In conclusion, PGE2 biosynthesis may represent a mechanism by which inflammatory macrophages protect themselves against the cytotoxic effects of NO.     

Parallel induction of nitric oxide and tetrahydrobiopterin synthesis by cytokines in rat glial cells

Activation of monocyte-derived macrophages with cytokines leads to the induction of nitric oxide synthase. Much less is known about the effects of cytokines on microglia, resident brain macrophages, or on astrocytes. In this study, we compared the induction by lipopolysaccharide, interferon-gamma, and tumor necrosis factor-alpha of nitric oxide production and synthesis of tetrahydrobiopterin, the required cofactor for nitric oxide synthase, in microglia and peritoneal macrophages. Activation of microglia induced parallel increases in nitric oxide and intracellular tetrahydrobiopterin levels, although induction of the latter appears to be somewhat more sensitive to diverse stimulators. As with macrophages, inducible nitric oxide production in microglia was blocked by inhibitors of tetrahydrobiopterin biosynthesis. Interleukin-2, an important component of the neuroimmunomodulatory system, was only a weak activator of microglia by itself but potently synergized with interferon-gamma to stimulate production of both nitric oxide and tetrahydrobiopterin. Astrocytes were also activated by lipopolysaccharide and combinations of cytokines but showed a somewhat different pattern of responses than microglia. Biopterin synthesis was increased to higher levels in astrocytes than in microglia, but maximal induction of nitric oxide production required higher concentrations of cytokines than microglia and the response was much lower. These results suggest that tetrahydrobiopterin synthesis in glial cells is a potential target for therapeutic intervention in acute CNS infections whose pathology may be mediated by overproduction of nitric oxide.     

Nitric oxide is overproduced by peritoneal macrophages in rat taurocholate pancreatitis: the mechanism of inducible nitric oxide synthase expression

To investigate the pathobiology of severe acute pancreatitis, we studied the expression of inducible nitric oxide synthase (iNOS) in peritoneal macrophages of experimental pancreatitis. Taurocholate (TCA) pancreatitis and cerulein (CE) pancreatitis were used as models of lethal and self-limited pancreatitis, respectively, and the mechanism of iNOS expression in peritoneal macrophages was studied. Serum nitrate and nitrite (NOx) concentrations increased during the course of TCA pancreatitis, and iNOS-immunoreactivity was detected in the peritoneal macrophages 12 h after the induction of TCA pancreatitis, but these phenomena were not observed in CE pancreatitis. Despite the difference in the iNOS expression, the iNOS messenger RNA (mRNA) and the activation of nuclear factor-kappa B (NF-kappa B) were detected in the peritoneal macrophages of both pancreatitis models. The supernatant of TCA pancreatitis ascites could induce iNOS in the peritoneal macrophages of normal rats in vitro, but the peritoneal lavage fluid of CE pancreatitis rats could not. The results indicated that there may be qualitative or quantitative differences in the macrophage activation between the two types of experimental pancreatitis and suggested that the ascites of rats with lethal acute pancreatitis contains some soluble factors that activate the macrophage/monocyte system and cause an overproduction of NO by the iNOS expression.     

Synergistic induction of DNA strand breakage caused by nitric oxide together with catecholamine: implications for neurodegenerative disease

Oxidative damage in neuronal cells and DNA has been implicated in the pathogenesis of various neurodegenerative diseases. We have demonstrated that DNA strand breakage is induced synergistically when plasmid DNA is incubated in the presence of both an NO-releasing compound (diethylamine NONOate, spermine NONOate, sodium nitroprusside) and a catecholamine (e.g., L-DOPA, dopamine, etc.). Either an NO-releasing compound or a catecholamine alone induced much fewer strand breaks. Tyrosine and tyramine as well as O-methylated derivatives of DOPA and dopamines did not exert this synergistic effect in the presence of NO. The DNA strand breakage induced by NO plus dopamine was inhibited by carboxy-PTIO (a trapping agent of NO and possibly other radicals), superoxide dismutase, and antioxidants such as N-acetylcysteine and ascorbate but not by HO. scavengers such as dimethyl sulfoxide, ethanol, and D-mannitol. These results suggest that the free HO. is not involved; rather a new oxidant(s) formed by the reaction between NO and catecholamine could be responsible for causing the DNA strand breakage. We propose that one of the responsible compounds is peroxynitrite (ONOO-), which is a strong oxidant and nitrating agent formed by the reaction between NO and O2.-. NO has been shown to oxidize catecholamines to form quinone derivatives, which lead to the generation of O2.- by the quinone/hydroquinone redox system. O2.- then reacts rapidly with NO to form peroxynitrite. However, it is also possible that other compounds such as NOx generated from catecholamines and NO may cause DNA damage. Our results implicate a synergistic interaction of catecholamines formed in dopaminergic neurons and NO formed by microglia or astrocytes or the two compounds produced within the same neuronal cells to produce a potent oxidant(s) which could cause damage in cells and DNA, thus playing an important role in the pathogenesis of various neurodegenerative diseases.