CAT2-mediated L-arginine transport and nitric oxide production in activated macrophages.
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ABSTRACT:
SUBMITTER: Kakuda DK
PROVIDER: S-EPMC1220283 | biostudies-other | 1999 Jun
REPOSITORIES: biostudies-other
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Project description:Transport of L-arginine and nitrite production were examined in the murine macrophage cell line J774. Bacterial lipopolysaccharide (LPS) induced a dose- and time-dependent stimulation of nitrite production, which was further increased in the presence of interferon-gamma. Nitrite synthesis was absolutely dependent on extracellular L-arginine and inhibited in the presence of L-lysine or L-ornithine. In unactivated J774 cells L-arginine transport was saturable, with an apparent Km of 0.14 +/- 0.04 mM and Vmax. of 15 +/- 2 nmol/h per 10(6) cells. LPS (1 microgram/ml) induced a time-dependent stimulation of L-arginine transport, and after 24 h the Vmax. increased to 34 +/- 2 nmol/h per 10(6) cells. These findings indicate that activation of J774 cells with LPS produces an increase in both L-arginine transport and nitrite synthesis. The elevated rate of L-arginine transport in activated J774 cells may provide a mechanism for sustained substrate supply during enhanced utilization of L-arginine for the generation of NO.
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Project description:Activated mouse macrophages metabolize arginine via NO synthase (NOS2) to produce NO as an antimicrobial effector. Published gene expression datasets provide little support for the activation of this pathway in human macrophages. Generation of NO requires the coordinated regulation of multiple genes. We have generated RNA-sequencing data from bone marrow-derived macrophages from representative rodent (rat), monogastric (pig and horse), and ruminant (sheep, goat, cattle, and water buffalo) species, and analyzed the expression of genes involved in arginine metabolism in response to stimulation with LPS. In rats, as in mice, LPS strongly induced Nos2, the arginine transporter Slc7a2, arginase 1 (Arg1), GTP cyclohydrolase (Gch1), and argininosuccinate synthase (Ass1). None of these responses was conserved across species. Only cattle and water buffalo showed substantial NOS2 induction. The species studied also differed in expression and regulation of arginase (ARG2, rather than ARG1), and amino acid transporters. Variation between species was associated with rapid promoter evolution. Differential induction of NOS2 and ARG2 between the ruminant species was associated with insertions of the Bov-A2 retrotransposon in the promoter region. Bov-A2 was shown to possess LPS-inducible enhancer activity in transfected RAW264.7 macrophages. Consistent with a function in innate immunity, NO production and arginine metabolism vary greatly between species and differences may contribute to pathogen host restriction.
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Project description:Macrophages play an important role in immune and inflammatory responses, and have been extensively studied in vitro using culture media such as RPMI1640 medium, Dulbecco's modified Eagle medium (DMEM), and Ham's F-12 medium (F-12). We found that the activation phenotypes of a murine macrophage-like cell line, J774.1/JA-4, were obviously different in two distinct culture media (F-12 and DMEM), both of which were supplemented with 10% of the same fetal bovine serum (FBS). Among these phenotypes, nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression, induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), were remarkably different. iNOS expression was higher in the macrophages cultured in DMEM than in F-12 for 20 h, while no significant differences were shown in NO production between in F-12 and DMEM. It might be the reason why DMEM have reduced NO production by the induced iNOS. Besides, [Formula: see text]-generating activity, and production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the activated macrophages were also different between the cultures in F-12 and DMEM. These results suggest that F-12 and DMEM contain certain components responsible for modification of macrophage activation processes and/or macrophage functions. Our present results provide evidence that the choice of culture medium is important in the study and analysis of macrophage activation.
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Project description:Mycobacterium tuberculosis is an intracellular pathogen of macrophages and escapes the macrophages' bactericidal effectors by interfering with phagosome-lysosome fusion. IFN-? activation renders the macrophages capable of killing intracellular mycobacteria by overcoming the phagosome maturation block, nutrient deprivation and exposure to microbicidal effectors including nitric oxide (NO). While the importance about NO for the control of mycobacterial infection in murine macrophages is well documented, the underlying mechanism has not been revealed yet. In this study we show that IFN-? induced apoptosis in mycobacteria-infected macrophages, which was strictly dependent on NO. Subsequently, NO-mediated apoptosis resulted in the killing of intracellular mycobacteria independent of autophagy. In fact, killing of mycobacteria was susceptible to the autophagy inhibitor 3-methyladenine (3-MA). However, 3-MA also suppressed NO production, which is an important off-target effect to be considered in autophagy studies using 3-MA. Inhibition of caspase 3/7 activation, as well as NO production, abolished apoptosis and elimination of mycobacteria by IFN-? activated macrophages. In line with the finding that drug-induced apoptosis kills intracellular mycobacteria in the absence of NO, we identified NO-mediated apoptosis as a new defense mechanism of activated macrophages against M. tuberculosis.
| S-EPMC3085516 | biostudies-literature
Project description:Immunosuppressive molecules within the aqueous humor (AqH) are thought to preserve ocular immune privilege by inhibiting proinflammatory NO production by macrophages (MΦs). Consistent with previous observations, we observed that although MΦs stimulated in the presence of AqH expressed NO synthase 2 (NOS2) protein, nitrite concentrations in culture supernatants, an indirect measure of NO production, did not increase. Interestingly, NOS2 enzymatic activity, as measured by the conversion of L-arginine (L-Arg) into L-citrulline, was augmented in lysates of MΦs stimulated in the presence of AqH. These data suggested that intracellular L-Arg may have been limited by AqH. However, we observed increased mRNA expression of the L-Arg transporter, cationic amino acid transporter 2B, and increased L-Arg uptake in MΦs stimulated in the presence of AqH. Arginases were expressed by stimulated Ms, but competition for L-Arg with NOS2 was excluded. Expression of GTP cyclohydrolase, which produces tetrahydrobiopterin (H(4)B), an essential cofactor for NOS2 homodimerization, increased after M stimulation in the presence or absence of AqH and NOS2 homodimers formed. Taken together, these data provided no evidence for inhibited NOS2 enzymatic activity by AqH, suggesting that a factor within AqH may have interfered with the measurement of nitrite. Indeed, we observed that nitrite standards were not measurable in the presence of AqH, and this effect was due to ascorbate in AqH. Controlling for interference by ascorbate revealed that AqH augmented NO production in MΦs via ascorbate, which limited degradation of H(4)B. Therefore, AqH may augment NO production in macrophages by stabilizing H(4)B and increasing intracellular L-Arg.
| S-EPMC3538947 | biostudies-literature
Project description:ImportanceIn October 2022, Mucorales fungi were listed in the "High Priority Group" on the first-ever list of fungal priority pathogens by the World Health Organization. As the causative agent of mucormycosis, Mucorales have become of great clinical and public health importance with growing mucormycosis numbers, notably with the exponential rise of COVID-19-associated mucormycosis cases. Despite the dire need, there are limited therapeutic options to treat mucormycosis. Our research fills in critical gaps of knowledge about how Mucorales fungi evade the host immune system. Specifically, we offer evidence that Mucorales block nitric oxide production, which is a key mediator and signaling molecule of the mammalian innate immune response to microbial pathogens. Our work offers new insight into immune evasion mechanisms by Mucorales fungi.
| S-EPMC10790689 | biostudies-literature
Project description:Previous studies have established that constitutive calcium-dependent ('low-output') nitric oxide synthase (NOS) is regulated by oxygen tension. We have investigated the role of oxygen tension in the synthesis of NO by the 'high-output' calcium-independent NOS in activated macrophages. Hypoxia increased macrophage NOS gene expression in the presence of one additional activator, such as lipopolysaccharide or interferon-gamma, but not in the presence of both. Hypoxia markedly reduced the synthesis of NO by activated macrophages (as measured by accumulation of nitrite and citrulline), such that, at 1% oxygen tension, NO accumulation was reduced by 80-90%. The apparent K(m) for oxygen calculated from cells exposed to a range of oxygen tensions was found to be 10.8%, or 137 microM, O(2) This value is considerably higher than the oxygen tension in tissues, and is virtually identical to that reported recently for purified recombinant macrophage NOS. The decrease in NO synthesis did not appear to be due to diminished arginine or cofactor availability, since arginine transport and NO synthesis during recovery in normoxia were normal. Analysis of NO synthesis during hypoxia as a function of extracellular arginine indicated that an altered V(max), but not K(m)(Arg), accounted for the observed decrease in NO synthesis. We conclude that oxygen tension regulates the synthesis of NO in macrophages by a mechanism similar to that described previously for the calcium-dependent low-output NOS. Our data suggest that oxygen tension may be an important physiological regulator of macrophage NO synthesis in vivo.
| S-EPMC1221301 | biostudies-other
Project description:BACKGROUND:Plasmodium falciparum haemozoin, a detoxification product of digested haemoglobin from infected erythrocytes, is released into the bloodstream upon schizont rupture and accumulates in leukocytes. High levels of haemozoin correlate with disease severity. Some studies have shown that concentrations of the substrate of inducible nitric oxide synthase (iNOS), L-arginine, as well as nitric oxide are low in patients infected with P. falciparum malaria. The present study investigates, in vitro, the role of P. falciparum haemozoin on nitric oxide production, iNOS expression in macrophages, and the possible interaction between L-arginine and haemozoin. METHODS:Plasmodium falciparum haemozoin was obtained from in vitro cultures through magnetic isolation. Phagocytosis of haemozoin by immortalized bone marrow derived macrophages was detected by confocal reflection combined with fluorescence microscopy. Nitrite concentrations in the supernatants was evaluated by Griess assay as a standard indication of nitric oxide production, while iNOS expression was detected on cell extracts by western blotting. Detection of L-arginine in haemozoin-treated or untreated media was achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS:Haemozoin synergizes in vitro with interferon-gamma to produce nitric oxide. However, when mouse macrophages were stimulated with haemozoin, a proportional increase of nitric oxide was observed up to 25 ?M of haemozoin, followed by a decrease with doses up to 100 ?M, when nitric oxide release was completely abrogated. This was not due to reactive oxygen species production, nor to an effect on iNOS activity. Interestingly, when at 24 h, haemozoin-treated macrophages were washed and incubated in fresh medium for further 24 h, the nitric oxide production was restored in a dose-response manner. Similar results were seen when L-arginine-enriched media was used in the stimulation. Moreover, muramyldipeptide, a strong nitric oxide inducer, was unable to activate macrophages to release nitric oxide in the presence of haemozoin-treated medium. By LC-MS/MS a complete depletion of L-arginine was observed in this haemozoin-treated, conditioned medium. CONCLUSIONS:It is proposed that haemozoin interacts with L-arginine reducing its availability for iNOS, and thus decreasing nitric oxide production. The clinical (or pathological) implications of these results are discussed.
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Project description:Nitric oxide (NO) is a key signaling molecule in plants, regulating a wide range of physiological processes. However, its origin in plants remains unclear. It can be generated from nitrite through a reductive pathway, notably via the action of the nitrate reductase (NR), and evidence suggests an additional oxidative pathway, involving arginine. From an initial screen of potential Arabidopsis thaliana mutants impaired in NO production, we identified copper amine oxidase 8 (CuAO8). Two cuao8 mutant lines displayed a decreased NO production in seedlings after elicitor treatment and salt stress. The NR-dependent pathway was not responsible for the impaired NO production as no change in NR activity was found in the mutants. However, total arginase activity was strongly increased in cuao8 knockout mutants after salt stress. Moreover, NO production could be restored in the mutants by arginase inhibition or arginine addition. Furthermore, arginine supplementation reversed the root growth phenotype observed in the mutants. These results demonstrate that CuAO8 participates in NO production by influencing arginine availability through the modulation of arginase activity. The influence of CuAO8 on arginine-dependent NO synthesis suggests a new regulatory pathway for NO production in plants.
| S-EPMC5447880 | biostudies-literature
Project description:L-Arginine itself and its metabolite-nitric oxide play great roles in intestinal physiology. However, the molecular mechanism underlying nitric oxide pathway regulating L-Arginine transport and cell growth is not yet fully understood. We report that inhibition of nitric oxide synthase (NOS) significantly induced cell apoptosis (p < 0.05), and promoted the rate of Arginine uptake and the expressions of protein for CAT-2 and y+LAT-1 (p < 0.05), while reduced protein expression of CAT-1. And NOS inhibition markedly decreased the activation of mammalian target of rapamycin (mTOR) and PI3K-Akt pathways by Arginine in the IPEC-1 cells (p < 0.05). Taken together, these data suggest that inhibition of NO pathway by L-NAME induces a negative feedback increasing of Arginine uptake and CAT-2 and y+LAT-1 protein expression, but promotes cell apoptosis which involved inhibiting the activation of mTOR and PI3K-Akt pathways.
| S-EPMC5444718 | biostudies-literature