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Results for “ARGINASE”

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PubMed1997

The glucocorticoid-responsive gene cascade. Activation of the rat arginase gene through induction of C/EBPbeta.

The gene for liver-type arginase, an ornithine cycle enzyme, is induced by glucocorticoids in a delayed secondary manner. An enhancer element located around intron 7 of the rat arginase gene shows delayed glucocorticoid responsiveness, and it harbors two sites binding with members of the CCAAT/enhancer binding protein (C/EBP) family. Here, we investigate the role of these C/EBP binding sites in glucocorticoid response of the arginase gene. When inserted in front of the herpes simplex virus thymidine kinase promoter, these C/EBP sites exhibited glucocorticoid responsiveness in reporter transfection assay using rat hepatoma H4IIE cells. In footprint analysis using nuclear extracts of H4IIE cells, profiles of the protected areas of the two C/EBP sites changed when cells were treated with dexamethasone. In gel shift analysis, the complex formation for the two C/EBP sites was augmented in response to dexamethasone. Antibody supershift/inhibition analysis demonstrated that a major portion of the binding proteins induced by dexamethasone is C/EBPbeta. Induction of arginase mRNA by dexamethasone was preceded by augmentation of the C/EBP site-binding activities, which followed increase in C/EBPbeta mRNA. These results were consistent with the notion that the glucocorticoid response of the arginase gene is mediated by C/EBPbeta.

Animals↗

PubMed2003

Yeast epiarginase regulation, an enzyme-enzyme activity control: identification of residues of ornithine carbamoyltransferase and arginase responsible for enzyme catalytic and regulatory activities.

In the presence of ornithine and arginine, ornithine carbamoyltransferase (OTCase) and arginase form a one-to-one enzyme complex in which the activity of OTCase is inhibited whereas arginase remains catalytically active. The mechanism by which these nonallosteric enzymes form a stable complex triggered by the binding of their respective substrates raises the question of how such a cooperative association is induced. Analyses of mutations in both enzymes identify residues that are required for their association, some of them being important for catalysis. In arginase, two cysteines at the C terminus of the protein are crucial for its epiarginase function but not for its catalytic activity and trimeric structure. In OTCase, mutations of putative ornithine binding residues, Asp-182, Asn-184, Asn-185, Cys-289, and Glu-256 greatly reduced the affinity for ornithine and impaired the interaction with arginase. The four lysine residues located in the SMG loop, Lys-260, Lys-263, Lys-265, and Lys-268, also play an important role in mediating the sensitivity of OTCase to ornithine and to arginase and appear to be involved in transducing and enhancing the signal given by ornithine for the closure of the catalytic domain.

Amino Acid Sequence↗

PubMed2005

The role of mitochondrially bound arginase in the regulation of urea synthesis: studies with [U-15N4]arginine, isolated mitochondria, and perfused rat liver.

The main goal of the current study was to elucidate the role of mitochondrial arginine metabolism in the regulation of N-acetylglutamate and urea synthesis. We hypothesized that arginine catabolism via mitochondrially bound arginase augments ureagenesis by supplying ornithine for net synthesis of citrulline, glutamate, N-acetylglutamate, and aspartate. [U-(15)N(4)]arginine was used as precursor and isolated mitochondria or liver perfusion as a model system to monitor arginine catabolism and the incorporation of (15)N into various intermediate metabolites of the urea cycle. The results indicate that approximately 8% of total mitochondrial arginase activity is located in the matrix, and 90% is located in the outer membrane. Experiments with isolated mitochondria showed that approximately 60-70% of external [U-(15)N(4)]arginine catabolism was recovered as (15)N-labeled ornithine, glutamate, N-acetylglutamate, citrulline, and aspartate. The production of (15)N-labeled metabolites was time- and dose-dependent. During liver perfusion, urea containing one (U(m+1)) or two (U(m+2)) (15)N was generated from perfusate [U-(15)N(4)]arginine. The output of U(m+2) was between 3 and 8% of total urea, consistent with the percentage of activity of matrix arginase. U(m+1) was formed following mitochondrial production of [(15)N]glutamate from [alpha,delta-(15)N(2)]ornithine and transamination of [(15)N]glutamate to [(15)N]aspartate. The latter is transported to cytosol and incorporated into argininosuccinate. Approximately 70, 75, 7, and 5% of hepatic ornithine, citrulline, N-acetylglutamate, and aspartate, respectively, were derived from perfusate [U-(15)N(4)]arginine. The results substantiate the hypothesis that intramitochondrial arginase, presumably the arginase-II isozyme, may play an important role in the regulation of hepatic ureagenesis by furnishing ornithine for net synthesis of N-acetylglutamate, citrulline, and aspartate.

Animals↗

PubMed2002

Arginase activity is inhibited by L-NAME, both in vitro and in vivo.

The present study investigated the ability of the arginine analog L-NAME (N(omega)-Nitro-L-arginine methyl ester) to modulate the activity of arginase. L-NAME inhibited the activity of arginase in lysates from rat colon cancer cells and liver. It also inhibited the arginase activity of tumor cells in culture. Furthermore, in vivo treatment of rats with L-NAME inhibited arginase activity in tumor nodules and liver, and the effect persisted after treatment ceased. The effect of L-NAME on arginase requires consideration when it is used in vivo in animal models with the aim of inhibiting endothelial NO-synthase, another enzyme using arginine as substrate.

Animals↗

PubMed1999

Effects of vanadate on expression of liver arginase in experimental diabetic rats.

This work was carried out to study the effects of vanadate on the expression of liver-type arginase in experimentally induced diabetes in the rat. The results showed that the activity and mRNA levels of arginase were increased significantly in the diabetic condition. Vanadate treatment reversed the increased activity and restored mRNA levels of arginase almost to the control values. The reversal effects of vanadate were found to be similar to those of insulin, and this further confirms the insulin-like effects of vanadate. ELISA and slot-blot assay observations were consistent with the biochemical measurements of enzyme activity. The increase in arginase activity and mRNA levels in diabetes and the decrease in insulin- and vanadate-treated animals may be due to the transcriptional regulation of arginase.

Animals↗

PubMed2001

The inhibition of arginase by N(omega)-hydroxy-l-arginine controls the growth of Leishmania inside macrophages.

Polyamine synthesis from l-ornithine is essential for Leishmania growth. We have investigated the dependence of Leishmania infection on arginase, which generates l-ornithine, in macrophages from BALB/c, C57BL/6, and nitric oxide synthase II (NOS II)-deficient mouse strains. We have found that N(omega)-hydroxy-l-arginine (LOHA), a physiological inhibitor of arginase, controls cellular infection and also specifically inhibits arginase activity from Leishmania major and Leishmania infantum parasites. The effect was proportional to the course of infection, concentration dependent up to 100 microM, and achieved without an increase in nitrite levels of culture supernatants. Moreover, when the l-arginine metabolism of macrophages is diverted towards ornithine generation by interleukin 4-induced arginase I, parasite growth is promoted. This effect can be reversed by LOHA. Inhibition of NOS II by N(G)-methyl-l-arginine (LNMMA) restores the killing obtained in the presence of interferon (IFN)-gamma plus lipolysaccharide (LPS), whereas the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-3-oxide-1-oxyl (carboxy-PTIO) was without effect. However, exogenous l-ornithine almost completely inhibits parasite killing when added in the presence of LOHA to macrophages from NOS II-deficient mice or to BALB/c-infected cells activated with IFN-gamma plus LPS. These results suggest that LOHA is an effector molecule involved in the control of Leishmania infection. In addition, macrophage arginase I induction by T helper cell type 2 cytokines could be a mechanism used by parasites to spread inside the host.

Animals↗

PubMed1979

The relationship of plasma arginine and kidney arginase activity to arginine degradation in chickens.

Experiments were carried out to study urea excretion during arginine or ornithine infusion into wing veins of hens previously fed diets that induced different arginase levels in their kidneys. Urea excretion was found to increase as plasma arginine increased. Hens with high levels of arginase activity in their kidneys had a greater increase in urea excretion than hens with low kidney arginase activity. Arginine degradation was also dependent on both the kidney arginase activity and on the plasma level of arginine. Ornithine infusion did not inhibit urea excretion even when high levels of plasma ornithine were reached. Even though ornithine was an in vitro inhibitor of arginase, no evidence was obtained of in vivo inhibition.

Animals↗

PubMed2004

Structure and function of arginases.

The arginases catalyze the divalent cation dependent hydrolysis of L-arginine to produce L-ornithine and urea. Although traditionally considered in terms of its role as the final enzyme of the urea cycle, the enzyme is found in a variety of nonhepatic tissues. These findings suggest that the enzyme may have other functions in addition to its role in nitrogen metabolism. High-resolution crystal structures have been determined for recombinant rat liver (type I) arginase and for recombinant human kidney (type II) arginase, their variants, and complexes with products and inhibitors. Each identical subunit of the trimeric enzyme contains an active site that lies at the bottom of a 15 A deep cleft. The 2 essential Mn(II) ions are located at the bottom of this cleft, separated by approximately 3.3 A and bridged by oxygens derived from 2 aspartic acid residues and a solvent-derived hydroxide. This metal bridging hydroxide is proposed to be the nucleophile that attacks the guanidinium carbon of substrate arginine. On the basis of this proposed mechanism, boronic acid inhibitors of the enzyme have been synthesized and characterized kinetically and structurally. These inhibitors display slow-onset inhibition at the pH optimum of the enzyme, and are found as tetrahedral species at the active site, as determined by X-ray diffraction. The potent inhibition of arginases I and II by these compounds has not only delineated key enzyme-substrate interactions, but has also led to a greater understanding of the role of arginase in nonhepatic tissues.

Animals↗

PubMed1990

Rat liver arginase suppresses mixed lymphocyte reaction.

An inhibitory factor to mixed lymphocyte reaction (MLR) was purified from the supernatant of rat liver homogenate by procedures including chloroform treatment, ammonium sulfate precipitation, ion-exchange column chromatography, and gel filtration. The molecular weight of the purified inhibitor was 34,000 on SDS-PAGE. We determined the amino acid sequence of the N-terminal region of the purified inhibitor to be Glu-Glu-Pro-Trp-Met-Ser-Met-Ser-Ser-Lys-Pro-Lys-Pro-Ile-Glu-. This sequence shows a high homology to a rat arginase, the amino acid sequence of which was predicted from the nucleic acid sequence of cloned rat arginase cDNA. The amino acid sequence of the purified arginase is 6 amino acid residues longer than the predicted one. The purified MLR inhibitor showed a high arginase activity. The inhibition mechanism was studied and it was discovered that L-arginine was depleted in the culture medium, and that the supply of L-arginine to the cell culture caused recovery of the incorporation of tritium thymidine. Here we present evidence that the MLR inhibitor from rat liver homogenate is the liver arginase. It is noteworthy that immune response may be controlled by a liver factor.

Amino Acid Sequence↗

PubMed2004

Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH), a fatal disease of unknown etiology characterized by impaired regulation of pulmonary hemodynamics and vascular growth, is associated with low levels of pulmonary nitric oxide (NO). Based upon its critical role in mediating vasodilation and cell growth, decrease of NO has been implicated in the pathogenesis of PAH. We evaluated mechanisms for low NO and pulmonary hypertension, including NO synthases (NOS) and factors regulating NOS activity, i.e. the substrate arginine, arginase expression and activity, and endogenous inhibitors of NOS in patients with PAH and healthy controls. PAH lungs had normal NOS I-III expression, but substrate arginine levels were inversely related to pulmonary artery pressures. Activity of arginase, an enzyme that regulates NO biosynthesis through effects on arginine, was higher in PAH serum than in controls, with high-level arginase expression localized by immunostaining to pulmonary endothelial cells. Further, pulmonary artery endothelial cells derived from PAH lung had higher arginase II expression and produced lower NO than control cells in vitro. Thus, substrate availability affects NOS activity and vasodilation, implicating arginase II and alterations in arginine metabolic pathways in the pathophysiology of PAH.

Arginase↗

PubMed2005

Pirfenidone inhibits lung allograft fibrosis through L-arginine-arginase pathway.

Transplant-related lung fibrosis is characterized by excessive fibro-collagenous deposition. Induction of arginase, an enzyme that metabolizes L-arginine to urea and L-ornithine, is vital for collagen synthesis. Pirfenidone is an investigational anti-fibrotic agent shown to be effective in blocking pulmonary fibrosis. The purpose of this study was to determine if pirfenidone was protective against the development of fibro-collagenous injury in rat lung orthotopic transplants through altering L-arginine-arginase metabolic pathways. Lung transplants were performed using Lewis donors and Sprague-Dawley recipients (allografts) or the same strain (isografts). Recipients were given pirfenidone (0.5% chow) 1-21-day post-transplantation. A significantly increased peak airway pressure (PawP) with excessive collagen deposition was found in untreated lung allografts. Pirfenidone treatment decreased PawP and collagen content in lung allografts. The beneficial effects were associated with downregulation of arginase protein expression and activity. In addition, pirfenidone decreased endogenous transforming growth factor (TGF)-beta level in lung allografts, and TGF-beta stimulated arginase activity in a dose-dependent manner in both lung tissue and fibroblasts. These results suggest that pirfenidone inhibits local arginase activity possibly through suppression of endogenous TGF-beta, hence, limiting the development of fibrosis in lung allografts.

Animals↗

PubMed2005

Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies. Alteration of substrate specificity by replacement of Asn149 with Asp.

To examine the interaction of human arginase II (EC 3.5.3.1) with substrate and manganese ions, the His120Asn, His145Asn and Asn149Asp mutations were introduced separately. About 53% and 95% of wild-type arginase activity were expressed by fully manganese activated species of the His120Asn and His145Asn variants, respectively. The K(m) for arginine (1.4-1.6 mM) was not altered and the wild-type and mutant enzymes were essentially inactive on agmatine. In contrast, the Asn149Asp mutant expressed almost undetectable activity on arginine, but significant activity on agmatine. The agmatinase activity of Asn149Asp (K(m) = 2.5 +/- 0.2 mM) was markedly resistant to inhibition by arginine. After dialysis against EDTA, the His120Asn variant was totally inactive in the absence of added Mn(2+) and contained < 0.1 Mn(2+).subunit(-1), whereas wild-type and His145Asn enzymes were half active and contained 1.1 +/- 0.1 Mn(2+).subunit(-1) and 1.3 +/- 0.1 Mn(2+).subunit(-1), respectively. Manganese reactivation of metal-free to half active species followed hyperbolic kinetics with K(d) of 1.8 +/- 0.2 x 10(-8) M for the wild-type and His145Asn enzymes and 16.2 +/- 0.5 x 10(-8) m for the His120Asn variant. Upon mutation, the chromatographic behavior, tryptophan fluorescence properties (lambda(max) = 338-339 nm) and sensitivity to thermal inactivation were not altered. The Asn149-->Asp mutation is proposed to generate a conformational change responsible for the altered substrate specificity of arginase II. We also conclude that, in contrast with arginase I, Mn(2+) (A) is the more tightly bound metal ion in arginase II.

Amino Acid Substitution↗

PubMed2005

Arginase I induction during Leishmania major infection mediates the development of disease.

In a previous work, we demonstrated that the induction of arginase I favored the replication of Leishmania inside macrophages. Now we have analyzed the differential expression of this enzyme in the mouse model of L. major infection. Ours results show that arginase I is induced in both susceptible and resistant mice during the development of the disease. However, in BALB/c-infected tissues, the induction of this protein parallels the time of infection, while in C57BL/6 mice, the enzyme is upregulated only during footpad swelling. The induction of the host arginase in both strains is mediated by the balance between interleukin-4 (IL-4) and IL-12 and opposite to nitric oxide synthase II expression. Moreover, inhibition of arginase reduces the number of parasites and delays disease outcome in BALB/c mice, while treatment with l-ornithine increases the susceptibility of C57BL/6 mice. Therefore, arginase I induction could be considered a marker of disease in leishmaniasis.

Animals↗

PubMed2003

Cruzipain, a major Trypanosoma cruzi antigen, promotes arginase-2 expression and survival of neonatal mouse cardiomyocytes.

An intense myocarditis is frequently found in the acute phase of Trypanosoma cruzi infection. Despite the cardiac damage, infected individuals may remain asymptomatic for decades. Thus T. cruzi may directly prevent cardiomyocyte death to keep heart destruction in check. Recently, it has been shown that Schwann cell invasion by T. cruzi, their prime target in the peripheral nervous system, suppressed host cell apoptosis caused by growth factor deprivation. Likewise, the trans-sialidase of T. cruzi reproduced this antiapoptotic activity of the parasite. In this study, we have investigated the effect of cruzipain, another important T. cruzi antigen, on survival and cell death of neonatal BALB/c mouse cardiomyocyte cultures. We have found that cruzipain, as well as T. cruzi infection, promoted survival of cardiomyocytes cultured under serum deprivation. The antiapoptotic effect was mediated by Bcl-2 expression but not by Bcl-xL expression. Because arginase activity is involved in cell differentiation and wound healing in most cell types and it favors parasite growth within the cell, we have further investigated the effect of cruzipain on the regulation of l-arginine metabolic pathways. Our results have revealed that cruzipain enhanced arginase activity and the expression of arginase-2 isoform but failed to induce nitric oxide synthase activity. In addition, the inhibition of arginase activity by NG-hydroxy-l-arginine, abrogated the antiapoptotic action of cruzipain. The results demonstrate that cruzipain may act as a survival factor for cardiomyocytes because it rescued them from apoptosis and stimulated arginase-2.

Animals↗

PubMed1998

Arginase modulates nitric oxide production in activated macrophages.

In macrophages and many other cell types, L-arginine is used as a substrate by both nitric oxide synthase (NOS) and arginase to produce nitric oxide (NO) and urea, respectively. Because the availability of L-arginine is a major determinant for NO synthesis in the activated macrophage, we hypothesized that NO production may be reduced by arginase via depleting the common substrate in this cell type. To test this hypothesis, we investigated the effect of an arginase inhibitor, L-norvaline, on NO production in J774A.1 mouse macrophages activated by lipopolysaccharide (LPS, 1.0 microgram/ml) for 22 h. In the absence of LPS, macrophages produced a low level of NO. In contrast, NO production from these cells was significantly increased in the presence of LPS. Increasing extracellular levels of L-arginine (0.01-0.8 mM) produced a concomitant increase in NO production of activated macrophages. L-Norvaline (10 mM), which specifically inhibits arginase activity (i.e., reducing urea production by 50%) without altering NOS activity, enhanced NO production (by 55%) from activated macrophages. The enhancement of NO production by L-norvaline was inversely related to the extracellular level of L-arginine. A more pronounced increase in NO production was observed at the lower level of extracellular L-arginine, i.e., a 55 vs. 28% increase for 0.05 and 0.1 mM extracellular L-arginine, respectively. When the L-arginine concentration exceeded 0.5 mM, the L-norvaline effect was abolished. These results indicate that arginase can compete with NOS for their common substrate and thus inhibit NO production. This regulatory mechanism may be particularly important when the extracellular supply of L-arginine is limited.

Animals↗

PubMed2005

Gene transfer with inducible nitric oxide synthase decreases production of urea by arginase in pulmonary arterial endothelial cells.

Nitric oxide (NO) is a vasodilator produced from L-arginine (L-Arg) by NO synthase (NOS). Gene therapy for hypertensive disorders has been proposed using the inducible isoform of NOS (iNOS). L-Arg also can be metabolized to urea and L-ornithine (L-Orn) by arginase, and L-Orn can be metabolized to proline and/or polyamines, which are vital for cellular proliferation. To determine the effect of iNOS gene transfer on arginase, we transfected bovine pulmonary arterial endothelial cells (bPAEC) with an adenoviral vector containing the gene for iNOS (AdiNOS). As expected, NO production in AdiNOS bPAEC was substantially greater than in control bPAEC. Although urea production was significantly less in the AdiNOS bPAEC than in the control bPAEC, despite similar levels of arginase I protein, AdiNOS transfection of bPAEC had no effect on the uptake of L-Arg. Inhibiting NO production with Nomega-nitro-L-arginine methyl ester increased urea production, and inhibiting urea production with L-valine increased nitrite production, in AdiNOS bPAEC. The addition of L-Arg to the medium increased urea production by AdiNOS bPAEC in a concentration-dependent manner. Thus, in these iNOS-transfected bPAEC, the transfected iNOS and native arginase compete for a common intracellular pool of L-Arg. This competition for substrate resulted in impaired proliferation in the AdiNOS-transfected bPAEC. These findings suggest that the use of iNOS gene therapy for pulmonary hypertensive disorders may not only be beneficial through NO-mediated pulmonary vasodilation but also may decrease vascular remodeling by limiting L-Orn production by native arginase.

Adenoviridae↗

PubMed2002

Activities of arginase I and II are limiting for endothelial cell proliferation.

Polyamines are essential for cell proliferation; therefore, we hypothesized that arginase I or arginase II activities, via production of ornithine for polyamine synthesis, may be limiting for proliferation of endothelial cells (EC). Bovine coronary venular EC stably transfected with a lacZ gene (lacZ-EC, control), rat arginase I cDNA (AI-EC), or mouse arginase II cDNA (AII-EC) were utilized to test this hypothesis. Cell-proliferation assays showed that EC proliferation was markedly increased in AI-EC and AII-EC compared with lacZ-EC. Expression of proliferating cell nuclear antigen was also enhanced in AI-EC and AII-EC. DL-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, was used to establish that increased polyamine synthesis was involved in mediating the enhanced growth of AI-EC and AII-EC. Addition of 5 mM DFMO to the culture medium completely abolished the differences in cellular putrescine concentrations and reduced the differences in spermidine concentrations among AI-EC, AII-EC, and lacZ-EC. The DFMO treatment also prevented an increase in AI-EC and AII-EC proliferation compared with lacZ-EC. Addition of 10 and 50 microM putrescine dose-dependently increased AI-EC, AII-EC, and lacZ-EC growth to the same extent. These results demonstrate that either arginase isoform can potentially play a role in modulating EC proliferation by regulating polyamine synthesis.

Animals↗

PubMed2002

Arginase inhibition slows the progression of renal failure in rats with renal ablation.

Exogenous arginine slows the progression of chronic renal failure (CRF) in remnant rats through a nitric oxide (NO)-dependent mechanism. We tested whether the inhibition of arginase could induce similar results through the increased availability of endogenous arginine. Three groups of remnant rats were studied for 8 wk: 1) untreated rats (REM); 2) remnant rats treated with 1% l-arginine (ARG); and 3) remnant rats administered a Mn(2+)-free diet to inhibit arginase (MNF). Normal rats (NOR) were used as controls. Liver arginase activity was depressed in MNF rats (-35% vs. REM, P < 0.01). No difference in metabolic data was detected among the groups throughout the study; blood pressure was significantly lower in MNF vs. ARG and REM rats after 6 wk (P < 0.001). The glomerular filtration rate (GFR) was greatly depressed in REM rats (-47% vs. NOR, P < 0.03) but was higher in ARG and MNF rats (+40 and +43% vs. REM, respectively, P < 0.05), with comparable changes in renal hemodynamics. Despite the better GFR, proteinuria was decreased in both ARG and MNF rats (-42%, P < 0.05, and -57%, P < 0.01, respectively, vs. REM rats). Arginine plasma levels, significantly reduced in REM rats (-41% vs. NOR, P < 0.01), were partially restored in MNF rats (+38% vs. REM), and urinary nitrite excretion, greatly depressed in REM rats (-76% vs. NOR, P < 0.01), was significantly increased in MNF rats (+209% vs. REM, P < 0.05). At the renal level, arginase activity was only slightly depressed in MNF rats (-18% vs. REM), but intrarenal concentrations of arginine were lower in this latter group (P < 0.05 vs. other groups). Beyond the hemodynamic modifications, MNF rats showed a lower glomerular sclerosis index (P < 0.05 vs. REM and ARG). Inhibition of arginase slows the progression of CRF in remnant rats similarly to arginine-treated rats; the better histological protection in MNF rats, however, suggests that additional factors are involved in these modifications.

Animals↗