Overexpression of arginase alters circulating and tissue amino acids and guanidino compounds and affects neuromotor behavior in mice.
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Arginine is an intermediate of the ornithine cycle and serves as a precursor for the synthesis of nitric oxide, creatine, agmatine and proteins. It is considered to be a conditionally essential amino acid because endogenous synthesis only barely meets daily requirements. In rapidly growing suckling neonates, endogenous arginine biosynthesis is crucial to compensate for the insufficient supply of arginine via the milk. Evidence is accumulating that the intestine rather than the kidney plays a major role in arginine synthesis in this period. Accordingly, ectopic expression of hepatic arginase in murine enterocytes by genetic modification induces a selective arginine deficiency. The ensuing phenotype, whose severity correlates with the level of transgene expression in the enterocytes, could be reversed with arginine supplementation. We analyzed the effect of arginine deficiency on guanidine metabolism and neuromotor behavior. Arginine-deficient transgenic mice continued to suffer from an arginine deficiency after the arginine biosynthetic enzymes had disappeared from the enterocytes. Postweaning catch-up growth in arginine-deficient mice was characterized by increased levels of all measured amino acids except arginine. Furthermore, plasma total amino acid concentration, including arginine, was significantly lower in adult male than in adult female transgenic mice. Decreases in the concentration of plasma and tissue arginine led to significant decreases in most metabolites of arginine. However, the accumulation of the toxic guanidino compounds, guanidinosuccinic acid and methylguanidine, corresponded inversely with circulating arginine concentration, possibly reflecting a higher oxidative stress under hypoargininemic conditions. In addition, hypoargininemia was associated with disturbed neuromotor behavior, although brain levels of toxic guanidino compounds and ammonia were normal. (+info)
Hepatocyte function in long-term organ culture of Amphimuma means liver.
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Fragments of liver from the adult urodele Amphiuma means, the Congo eel, were maintained in organ culture for up to 70 days. The normal electrophoretic patterns of several enzymes were retained. The activities of ornithine transcarbamylase, arginase, glutamate oxalacetate transaminase and glutamate pyruvate transaminase, and urea production, glucose uptake and tissue glycogen content remained relatively constant throughout the culture period. Histological organization and hepatocyte ultrastructure were also retained. Liver fragments survived better in media based on MEM or BME than in medium based on Leibovitz L15. Since many aspects of tissue-specific structure and function are retained, long-term amphibian organ culture is well suited to studies on the control of hepatocyte function and on the effects of metabolites, hormones, drugs and toxins. (+info)
A defect in HIV-1 transgenic murine macrophages results in deficient nitric oxide production.
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HIV transgenic mice bearing multiple copies of a noninfectious (Deltagag/pol) proviral DNA were tested for the systemic production of nitric oxide (NO). Serum levels of NO metabolites were reduced about 50% in HIV transgenic mice compared with nontransgenic sibling mice. This difference persisted when NO production was induced with peritoneal injections of bacterial endotoxin (LPS). Peritoneal inflammatory macrophages, but not resident peritoneal macrophages, derived from HIV-1 transgenic mice and activated in vitro with LPS and IFN-gamma (or tumor necrosis factor alpha and IFN-gamma) also produced about 50% less NO than did macrophages harvested from nontransgenic littermates. Isogenic, transgenic mice bearing mutated nef or vpr genes had normal serum levels of NO metabolites and their macrophages produced normal levels of NO when stimulated. An explanation for the reduced NO response of HIV[Vpr+Nef+] macrophages was not apparent from measured levels of iNOS expression, viral gene expression, or arginase activity in activated macrophages. Inhibition of nitric oxide synthase (NOS) isoforms with L-NAME or aminoguanidine blocked time-dependent increases in HIV gene expression in activated macrophages cultured ex vivo. Inhibition with L-NAME occurred despite high levels of NO generated by iNOS, and exogenously supplied NO induced HIV gene expression only weakly, suggesting that cNOS had the greater influence on proviral gene induction. This system is presented as a model of HIV-1 proviral gene expression and dysfunction in macrophages. (+info)
Involvement of food intake in the lysine-arginine antagonism in chicks.
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Studies were conducted to evaluate the involvement of food intake in the lysine-arginine antagonism. Diets were formulated to compensate for the metabolic consequences of excess dietary lysine; induction of renal arginase activity, depression of heptic glycine transamidinase, and urinary losses of arginine. This was accomplished by inclusion of creatine in the basal diet, use of a moderate excess of lysine that did not increase urinary arginine excretion, and addition of the arginase depressors, alpha-aminoisobutyric acid (AIB) and L-threonine, to diets containing excess lysine. When chicks were fed diets containing excess lysine ad libitum, growth and efficiency of arginine retention were reduced. Supplementation of the diets with AIB and threonine markedly reduced the growth depression and restored efficiency of arginine utilization. When chicks were force-fed the diet containing excess lysine, growth was depressed, and body composition was altered. Inclusion of AIB and threonine in the diet containing excess lysine resulted in growth and body composition equivalent to levels of force-fed controls. In a second experiment the basal diet and basal supplemented with AIB and threonine were pair-fed to lysine-supplemented diets containing AIB and threonine. Body weight gains and body composition of all groups were similar. In other experiments, food intake increased within 24 hours (P less than 0.05) and probably within 12 hours (P less than 0.10) after removal of excess lysine from the diet. It is concluded that a portion of the lysine-arginine antagonism is due to a primary effect of lysine on regulation of food intake. (+info)
Activation of caspase-3 activity and apoptosis in MDA-MB-468 cells by N(omega)-hydroxy-L-arginine, an inhibitor of arginase, is not solely dependent on reduction in intracellular polyamines.
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We have shown previously that (NOHA) an intermediate in the nitric oxide (NO) synthetic pathway and an inhibitor of arginase significantly reduced intracellular polyamines, activated caspase-3 and induced apoptosis in the human breast cancer cell line MDA-MB-468. These actions of NOHA were abolished in the presence of exogenous L-ornithine suggesting that a reduction in the intracellular polyamine content might be responsible for the activation of caspase-3 and apoptotic actions of NOHA. In order to further explore this possibility, we used SAM-486A and alpha-difluoromethylornithine (DFMO), which are inhibitors of S-adenosylmethionine decarboxylase (SAMDC), and ornithine decarboxylase (ODC), respectively, either alone or in combination to reduce the intracellular polyamine levels. We then assessed whether a reduction in polyamine levels by these two compounds to a similar degree to that produced by NOHA activated caspase-3 which occurs prior to the onset of apoptosis. We observed that both SAM-486A and DFMO, either alone or in combination, inhibited cell proliferation, induced p21 and arrested cells in the G(0)-G(1) phase of the cell cycle but failed to activate caspase-3 as assessed by enzymatic assay of caspase-3, western blot analysis of the proteolytic cleavage of caspase-3 protein as well as TUNEL assay. Furthermore, pre-incubation of the cells with SAM-486A and DFMO for 4 days, either alone or in combination significantly inhibited the activation of caspase-3 and apoptosis by NOHA when compared with that observed with cells treated with NOHA alone. Our results, therefore, indicate that the activation of caspase-3 and apoptosis observed with NOHA cannot be solely explained by a reduction in intracellular polyamine levels and that other mechanisms need to be also considered. (+info)
Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism.
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Type 2 cytokines regulate fibrotic liver pathology in mice infected with Schistosoma mansoni. Switching the immune response to a type 1-dominant reaction has proven highly effective at reducing the pathologic response. Activation of NOS-2 is critical, because type 1-deviated/NO synthase 2 (NOS-2)-deficient mice completely fail to control their response. Here, we demonstrate the differential regulation of NOS-2 and arginase type 1 (Arg-1) by type 1/type 2 cytokines in vivo and for the first time show a critical role for arginase in the pathogenesis of schistosomiasis. Using cytokine-deficient mice and two granuloma models, we show that induction of Arg-1 is type 2 cytokine dependent. Schistosome eggs induce Arg-1, while Mycobacterium avium-infected mice develop a dominant NOS-2 response. IFN-gamma suppresses Arg-1 activity, because type 1 polarized IL-4/IL-10-deficient, IL-4/IL-13-deficient, and egg/IL-12-sensitized animals fail to up-regulate Arg-1 following egg exposure. Notably, granuloma size decreases in these type-1-deviated/Arg-1-unresponsive mice, suggesting an important regulatory role for Arg-1 in schistosome egg-induced pathology. To test this hypothesis, we administered difluoromethylornithine to block ornithine-aminodecarboxylase, which uses the product of arginine metabolism, L-ornithine, to generate polyamines. Strikingly, granuloma size and hepatic fibrosis increased in the ornithine-aminodecarboxylase-inhibited mice. Furthermore, we show that type 2 cytokine-stimulated macrophages produce proline under strict arginase control. Together, these data reveal an important regulatory role for the arginase biosynthetic pathway in the regulation of inflammation and demonstrate that differential activation of Arg-1/NOS-2 is a critical determinant in the pathogenesis of granuloma formation. (+info)
L-Arginine is not the limiting factor for nitric oxide synthesis by human alveolar macrophages in vitro.
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Unlike murine mononuclear phagocytes, human macrophages do not release high amounts of nitric oxide (NO) in vitro despite the presence of nitric oxide synthase (NOS). To determine whether this limited NO synthesis in vitro is due to limited availability of the NOS substrate L-arginine, and putative NOS inhibiting factors present in foetal serum preparations, both alveolar macrophages (AM) and monocyte derived macrophages (MDM) were incubated in various circumstances. Nitrite production measured using stimulated AM was typically <5 pmol x min(-1) x 10(-6) cells. A range of stimuli were tested, but without result. Furthermore, incubation of MDMs with normal human serum or purified bovine serum albumin instead of foetal calf serum failed to enhance NO production. Moreover, neither the use of arginase inhibitors nor the addition of surplus L-arginine resulted in an increased NO synthesis. Interestingly, addition of the NOS intermediate Nomega-hydroxy-L-arginine (100 microM) to AM led to nitrite release, which was unaffected by the NOS inhibitor amino guanidine showing that this effect is NOS independent. It is concluded that the limited nitric oxide production of human macrophages in vitro can neither be explained by limited availability of L-arginine, nor by nitric oxide synthase inhibiting substances in foetal serum. Furthermore, it is shown that nitrite release from Nomega-hydroxy-L-arginine by alveolar macrophages is nitric oxide synthase independent. (+info)
Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: a strategy for bacterial survival.
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The antimicrobial effect of nitric oxide (NO) is an essential part of innate immunity. The vigorous host response to the human gastric pathogen Helicobacter pylori fails to eradicate the organism, despite up-regulation of inducible NO synthase (iNOS) in the gastric mucosa. Here we report that wild-type strains of H. pylori inhibit NO production by activated macrophages at physiologic concentrations of l-arginine, the common substrate for iNOS and arginase. Inactivation of the gene rocF, encoding constitutively expressed arginase in H. pylori, restored high-output NO production by macrophages. By using HPLC analysis, we show that l-arginine is effectively consumed in the culture medium by wild-type but not arginase-deficient H. pylori. The substantially higher levels of NO generated by macrophages cocultured with rocF-deficient H. pylori resulted in efficient killing of the bacteria, whereas wild-type H. pylori exhibited no loss of survival under these conditions. Killing of the arginase-deficient H. pylori was NO-dependent, because peritoneal macrophages from iNOS(-/-) mice failed to affect the survival of the rocF mutant. Thus, bacterial arginase allows H. pylori to evade the immune response by down-regulating eukaryotic NO production. (+info)