Macrophage arginine metabolism and the inhibition or stimulation of cancer.

The potential of the immune system to inhibit or stimulate tumor growth is a vivid example of the "two-edged sword" nature of immune responses. Our results provide evidence that this dual capacity can be attributed, in part, to the dual pathways of arginine metabolism exhibited by intratumor macrophages. Specifically, i.p. tumor rejection in P815-preimmunized mice is accompanied by an upshift in intratumor macrophage arginine metabolism to the nitric oxide (NO) synthase pathway that yields citrulline and NO. A rapid and marked local increase in IFN-gamma (both mRNA and protein) in preimmunized mice during tumor rejection suggests that this cytokine plays a role in up-regulating nitric oxide production in vivo. Unlike tumor rejection, progressive i.p. P815 tumor growth in naive mice is associated with a marked decline in the production of citruline/NO by intratumor macrophages. Examination of macrophage arginine metabolism via arginase revealed a pattern opposite that of NO synthase. The local production of ornithine/urea markedly increases during progressive tumor growth whereas arginase activity decreases during tumor rejection. Inasmuch as nitric oxide inhibits tumor cell replication whereas ornithine is the precursor of polyamines required for cell replication, these results are consistent with the conclusion that the pathway macrophages use to metabolize arginine can influence the type of host immune responses against cancer and other conditions.     

Cutting edge: Stat6-dependent substrate depletion regulates nitric oxide production

The cytokines IL-4 and IL-13 inhibit the production of NO from activated macrophages through an unresolved molecular mechanism. We show here that IL-4 and IL-13 regulate NO production through depletion of arginine, the substrate of inducible NO synthase (iNOS). Inhibition of NO production from murine macrophages stimulated with LPS and IFN-gamma by IL-4 or IL-13 was dependent on Stat6, cell density in the cultures, and pretreatment for at least 6 h. IL-4/IL-13 did not interfere with the expression or activity of iNOS but up-regulated arginase I (the liver isoform of arginase) in a Stat6-dependent manner. Addition of exogenous arginine completely restored NO production in IL-4-treated macrophages. Furthermore, impaired killing of the intracellular pathogen Toxoplasma gondii in IL-4-treated macrophages was overcome by supplementing L-arginine. The simple system of regulated substrate competition between arginase and iNOS has implications for understanding the physiological regulation of NO production.     

Inhibition of arginase ameliorates experimental ulcerative colitis in mice

Abstract

Nitric oxide (NO) is produced from the conversion of L-arginine by NO synthase (NOS) and regulates a variety of processes in the gastrointestinal tract. Considering the increased activity of arginase in colitis tissue, it is speculated that arginase could inhibit NO synthesis by competing for the same L-arginine substrate, resulting in the exacerbation of colitis. We examined the role of arginase and its relationship to NO metabolism in dextran sulfate sodium (DSS)-induced colitis. Experimental colitis was induced in mice by administration of 2.5% DSS in drinking water for 8 days. Treatment for arginase inhibition was done by once daily intraperitoneal injection of N^ω-hydroxy-nor- arginine (nor-NOHA). On day 8, we evaluated clinical parameters (body weight, disease activity index, and colon length), histological features, the activity and expression of arginase, L-arginine content, the expression of NO synthase (NOS), and the concentration of NO end-product (NOx: nitrite + nitrate). Administration of nor-NOHA improved the worsened clinical parameters and histological features in DSS-induced colitis. Treatment with nor-NOHA attenuated the increased activity of arginase, upregulation of arginase Ι at both mRNA and protein levels, and decreased the content of L-arginine in colonic tissue in the DSS-treated mice. Conversely, despite the decreased expression of NOS2 mRNA, the decreased concentration of NOx in colonic tissues was restored to almost normal levels. The consumption of L-arginine by arginase could lead to decreased production of NO from NOS, contributing to the pathogenesis of the colonic inflammation; thus, arginase inhibition might be effective for improving colitis.     

Enteral arginase II provides ornithine for citrulline synthesis

The synthesis of citrulline from arginine in the small intestine depends on the provision of ornithine. To test the hypothesis that arginase II plays a central role in the supply of ornithine for citrulline synthesis, the contribution of dietary arginine, glutamine, and proline was determined by utilizing multitracer stable isotope protocols in arginase II knockout (AII(-/-)) and wild-type (WT) mice. The lack of arginase II resulted in a lower citrulline rate of appearance (121 vs. 137 μmol·kg(-1)·h(-1)) due to a reduced availability of ornithine; ornithine supplementation was able to restore the rate of citrulline production in AII(-/-) to levels comparable with WT mice. There were significant differences in the utilization of dietary citrulline precursors. The contribution of dietary arginine to the synthesis of citrulline was reduced from 45 to 10 μmol·kg(-1)·h(-1) due to the lack of arginase II. No enteral utilization of arginine was observed in AII(-/-) mice (WT = 25 μmol·kg(-1)·h(-1)), and the contribution of dietary arginine through plasma ornithine was reduced in the transgenic mice (20 vs. 13 μmol·kg(-1)·h(-1)). Dietary glutamine and proline utilization were greater in AII(-/-) than in WT mice (20 vs. 13 and 1.4 vs. 3.7 μmol·kg(-1)·h(-1), respectively). Most of the contribution of glutamine and proline was enteral rather than through plasma ornithine. The arginase isoform present in the small intestinal mucosa has the role of providing ornithine for citrulline synthesis. The lack of arginase II results in a greater contribution of plasma ornithine and dietary glutamine and proline to the synthesis of citrulline.     

Cat2 L-arginine transporter-deficient fibroblasts can sustain nitric oxide production.

High-output nitric oxide (NO) production by nitric oxide synthase 2 (NOS2) contributes to normal cellular processes and pathophysiological conditions. The transport of L-arginine, the substrate for NOS2, is required for sustained NO production by NOS2. L-Arginine can be transported by several kinetically defined transport systems, although the majority of arginine uptake is mediated by transport system y(+), encoded by the Cat1-3 gene family. Using macrophages from Cat2-deficient mice, we previously determined that arginine uptake via CAT2 is absolutely required for sustained NO production. Because NO production by fibroblasts is important in wound healing, we sought to determine whether CAT2 is required for NO production in cytokine-stimulated Cat2-deficient and wild-type embryonic fibroblasts. Although macrophages and fibroblasts both required extracellular L-arginine for NO production, NO synthesis by activated Cat2(-/-) fibroblasts was reduced only 19%, whereas Cat2(-/-) macrophages were virtually unable to produce NO. As expected, activated Cat2(-/-) fibroblasts had reduced system y(+)-mediated arginine uptake. However, their reduced NO output was not the result of a significant difference in intracellular L-arginine levels following cytokine stimulation. Uptake experiments revealed that the L-arginine transport system y(+)L was the major cationic amino acid carrier in fibroblasts of both genotypes. We conclude that NO production in embryonic fibroblasts is only partially dependent on CAT2 and that other compensating transporters provide arginine for NOS2-mediated NO synthesis. The data demonstrate that fibroblasts and macrophages have differential dependence on CAT2-mediated L-arginine transport for NO synthesis. The important physiological implication of this finding is discussed.     

Ethanol inhibits L-arginine uptake and enhances NO formation in human placenta.

The acute effects of ethanol (20-60 mM) on L-arginine uptake and nitric oxide (NO) formation was investigated in human placental cotyledons perfused at constant flow. Ethanol (40 mM) decreased L-[3H]arginine uptake from 27.6 +/- 2.3 to 15.8 +/- 1.3 per cent (P < 0.05) of the injected dose and significantly enhanced NO levels in the perfusate from 0.88 +/- 0.11 to 2.80 +/- 0.39 microM. Ethanol also elicited the constriction of placental vessels. The effects of ethanol (20-60 mM) on L-arginine uptake and endothelial NO synthase (eNOS) activity were also investigated in cultured human umbilical vein endothelial cells (HUVEC). After 60 min of ethanol (40 mM) exposure, basal L-[3H]arginine uptake (4.7 +/- 0.3 pmol/microg protein/min) was inhibited by 60 per cent (P < 0.05). Basal eNOS activity in HUVEC determined under "no flow" (static) conditions was significantly increased (approximately 1.8 fold) by 60 mM ethanol. These data are consistent with a stimulatory effect of ethanol on eNOS activity in both basal and flow-stimulated conditions, which may serve a protective role against its vasoconstrictive acute effect. While acute ethanol administration inhibits L-arginine uptake, the present results do not allow us to speculate on the effects of chronic ethanol exposure on NO formation in the fetoplacental unity.     

The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and p38 mitogen-activated protein kinase in IL-13-mediated arginase I induction in macrophages: its implications in IL-13-inhibited nitric oxide production

In macrophages, L-arginine can be used by NO synthase and arginase to form NO and urea, respectively. Therefore, activation of arginase may be an effective mechanism for regulating NO production in macrophages through substrate competition. Here, we examined whether IL-13 up-regulates arginase and thus reduces NO production from LPS-activated macrophages. The signaling molecules involved in IL-13-induced arginase activation were also determined. Results showed that IL-13 increased arginase activity through de novo synthesis of the arginase I mRNA and protein. The activation of arginase was preceded by a transient increase in intracellular cAMP, tyrosine kinase phosphorylation, and p38 mitogen-activated protein kinase (MAPK) activation. Exogenous cAMP also increased arginase activity and enhanced the effect of IL-13 on arginase induction. The induction of arginase was abolished by a protein kinase A (PKA) inhibitor, KT5720, and was down-regulated by tyrosine kinase inhibitors and a p38 MAPK inhibitor, SB203580. However, inhibition of p38 MAPK had no effect on either the IL-13-increased intracellular cAMP or the exogenous cAMP-induced arginase activation, suggesting that p38 MAPK signaling is parallel to the cAMP/PKA pathway. Furthermore, the induction of arginase was insensitive to the protein kinase C and p44/p42 MAPK kinase inhibitors. Finally, IL-13 significantly inhibited NO production from LPS-activated macrophages, and this effect was reversed by an arginase inhibitor, L-norvaline. Together, these data demonstrate for the first time that IL-13 down-regulates NO production through arginase induction via cAMP/PKA, tyrosine kinase, and p38 MAPK signalings and underline the importance of arginase in the immunosuppressive activity of IL-13 in activated macrophages.     

Nitric oxide and L-arginine metabolism in a devascularized porcine model of acute liver failure

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.     

Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate

Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666].     

Induction of nitric oxide synthase is a necessary precondition for expression of tumor necrosis factor-independent tumoricidal activity by activated macrophages.

Various bacteria and bacterial products induce in pure, lymphocyte-free bone marrow-derived mononuclear phagocytes (BMM?) the generation of tumor necrosis factor, nitric oxide (NO) synthase, NO and nitrite (NO2-), the flow of L-arginine to citrulline, and tumoricidal activity. The flow of L-arginine to citrulline and formation of NO/NO2- on the one hand and expression of tumoricidal activity were not always closely related; however, these parameters were suppressed in a dose-dependent manner by the flavoprotein inhibitor, diphenyleneiodonium (DPI) and the L-arginine analogue, NG-monomethyl-L-arginine (NMMA). The findings support the concept of a central role of the NO synthase pathway in the generation of tumor necrosis factor-independent tumoricidal activity by activated macrophages but the exact conditions which enable the transfer of the lytic principle from the effector to the target cell remain to be elucidated.     

Regulation of nitric oxide production by arginine metabolic enzymes

Nitric oxide (NO) is synthesized from arginine by NO synthase (NOS), and the availability of arginine is one of the rate-limiting factors in cellular NO production. Citrulline, which is formed as a by-product of the NOS reaction, can be recycled to arginine by successive actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), forming the citrulline-NO cycle. AS and sometimes AL have been shown to be coinduced with inducible NOS (iNOS) in various cell types including activated macrophages, vascular smooth muscle cells, glial cells, neuronal PC12 cells, and pancreatic beta-cells. Cationic amino acid transporter (CAT)-2 is induced in activated macrophages but not in PC12 cells. On the other hand, arginase can downregulate NO production by decreasing intracellular arginine concentrations. iNOS and arginase activities are regulated reciprocally in macrophages by cytokines, and this may guarantee the efficient production of NO. In contrast, iNOS and arginase isoforms (type I and II) are coinduced in lipopolysaccharide (LPS)-activated macrophages. These results indicate that NO production is modulated by the uptake, recycling, and degradation of arginine.     

Arginine transport via cationic amino acid transporter 2 plays a critical regulatory role in classical or alternative activation of macrophages

Arginine is processed by macrophages in response to the cytokines to which these cells are exposed. Th1-type cytokines induce NO synthase 2, which metabolizes arginine into nitrites, while the Th2-type cytokines produce arginase, which converts arginine into polyamines and proline. Activation of bone marrow-derived macrophages by these two types of cytokines increases L-arginine transport only through the y(+) system. Analysis of the expression of the genes involved in this system showed that Slc7A1, encoding cationic amino acid transporters (CAT)1, is constitutively expressed and is not modified by activating agents, while Slc7A2, encoding CAT2, is induced during both classical and alternative activation. Macrophages from Slc7A2 knockout mice showed a decrease in L-arginine transport in response to the two kinds of cytokines. However, while NO synthase 2 and arginase expression were unmodified in these cells, the catabolism of arginine was impaired by both pathways, producing smaller amounts of nitrites and also of polyamines and proline. In addition, the induction of Slc7A2 expression was independent of the arginine available and of the enzymes that metabolize it. In conclusion, the increased arginine transport mediated by activators is strongly regulated by CAT2 expression, which could limit the function of macrophages.     

Glutamate Receptor Agonists Stimulate Nitric Oxide Synthase in Primary Cultures of Cerebellar Granule Cells

The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca(2+)-dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor-mediated activation of nitric oxide (NO) synthase. The NMDA-induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled L-arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose-dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK-801 and by the inhibitors of NO synthase, NG-monomethyl-L-arginine (MeArg) and NG-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 microM) stimulated 45Ca2+ influx to the same extent as 100 microM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.     

Radiochemical HPLC detection of arginine metabolism: measurement of nitric oxide synthesis and arginase activity in vascular tissue.

Nitric oxide (NO) plays a key role in vascular homeostasis. Accurate measurement of NO production by endothelial nitric oxide synthase (eNOS) is critical for the investigation of vascular disease mechanisms using genetically modified animal models. Previous assays of NO production measuring the conversion of arginine to citrulline have required homogenisation of tissue and reconstitution with cofactors including NADPH and tetrahydrobiopterin. However, the activity and regulation of NOS in vivo is critically dependant on tissue levels of these cofactors. Therefore, understanding eNOS regulation requires assays of NO production in intact vascular tissue that do not depend on the addition of exogenous cofactors and have sufficient sensitivity and specificity. We describe a novel technique, using radiochemical detection of arginine to citrulline conversion, to measure NO production within intact mouse aortas, without exogenous cofactors. We demonstrate the presence of arginase activity in mouse aortas which has the potential to confound this assay. Furthermore, we describe the use of N-hydroxy-nor-L-arginine (nor-NOHA) to inhibit arginase and permit specific detection of NO production in intact mouse tissue. Using this technique we demonstrate a 2.4-fold increase in NO production in aortas of transgenic mice overexpressing eNOS in the endothelium, and show that this technique has high specificity and high sensitivity for detection of in situ NO synthesis by eNOS in mouse vascular tissue. These results have important implications for the investigation of NOS regulation in cells and tissues.     

N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine.

Authentic N omega-hydroxy-L-arginine was synthesized and used to determine whether it is an intermediate in nitric oxide (.NO) synthesis from L-arginine by macrophage .NO synthase. The apparent Km (6.6 microM) and Vmax (99 nmol x min-1 x mg-1) observed with N omega-hydroxy-L-arginine were similar to those observed with L-arginine (Km = 2.3 microM; Vmax = 54 mumol x min-1 x mg-1). N omega-Hydroxy-D-arginine was not a substrate. Stable isotope studies showed that .NO synthase exclusively oxidized the hydroxylated nitrogen of N omega-hydroxy-L-arginine, forming .NO and L-citrulline. As with L-arginine, O2 was the source of the ureido oxygen in L-citrulline from N omega-hydroxy-L-arginine. In the presence of excess N omega-hydroxy-L-arginine, .NO synthase generated a metabolite of L-[14C]arginine that cochromatographed with authentic N omega-hydroxy-L-arginine. The labeled metabolite exhibited identical chromatographic behavior in three solvent systems and generated the same product (L-citrulline) upon alkaline hydrolysis as authentic N omega-hydroxy-L-arginine. Experiments were then run to identify which redox cofactor (NADPH or tetrahydrobiopterin) participated in the enzymatic synthesis of N omega-hydroxy-L-arginine. Both cofactors were required for synthesis of .NO from either N omega-hydroxy-L-arginine or L-arginine. However, with L-arginine, the synthesis of 1 mol of .NO was coupled to the oxidation of 1.52 +/- 0.02 mol of NADPH; whereas with N omega-hydroxy-L-arginine, only 0.53 +/- 0.04 mol of NADPH was oxidized per mol of .NO formed. These results support a mechanism in which N omega-hydroxy-L-arginine is generated as an intermediate in .NO synthesis through an NADPH-dependent hydroxylation of L-arginine.     

Indomethacin prevents the induction of inducible nitric oxide synthase in murine peritoneal macrophages and decreases their nitric oxide production

Indomethacin (0.14-.5 mM concentration) inhibits nitric oxide production in murine peritoneal macrophages. This was evidenced by measuring both nitrite production or 14C-L-citrulline formation. The inhibition was caused by the diminution of de novo inducible nitric oxide synthase production as demonstrated by Western blotting experiment. The effect of indomethacin after 4 h treatment was irreversible. NO synthase and arginase activities and the uptake of arginine were not directly affected by the drug. Indomethacin also decreased uridine incorporation in macrophages. The effect of indomethacin on the induction of other enzymes (i.e. arginase) was weaker.     

Arginase modulates Salmonella induced nitric oxide production in RAW264.7 macrophages and is required for Salmonella pathogenesis in mice model of infection

Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of l-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate l-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor Aminoguanidine reverts the attenuation in arginase-blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of l-arginine. The whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner.     

Inverse relation in the de novo arginase synthesis and nitric oxide production in murine and rat peritoneal macrophages in long-term cultures in vitro.

1. The de novo synthesis of arginase was much higher in murine than in rat peritoneal macrophages. This process was inhibited irreversibly by protein synthesis inhibitors and reversibly by glycolysis blockers. 2. Rat macrophages produce more nitric oxide (NO) than murine cells. NO production was inhibited by the inhibitors of protein synthesis or glycolysis. 3. The loading of macrophages by exogenous arginine for 24 hr in vitro resulted in the increase of arginase and nitrite in macrophages to different extents. 4. No great differences in lysozyme production was observed. 5. The proportion of arginine taken up and incorporated is contrasted in murine and rat macrophages.