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PubMed1997

Chromosomal localization of the human arginase II gene and tissue distribution of its mRNA.

Liver-type arginase (arginase I) is expressed almost exclusively in the liver and catalyzes the last step of urea synthesis, whereas the nonhepatic type (arginase II) is expressed in extrahepatic tissues and is probably involved in down-regulation of nitric oxide synthesis. We isolated cDNA for human arginase II (T. Gotoh et al., 1996, FEBS Lett. 395, 119-122). Fluorescence in situ hybridization mapping and PCR mapping studies with somatic cell hybrid panels and a radiation hybrid panel localized the arginase II gene to chromosome 14q24.1-24.3. Dot-blot analysis showed that arginase II mRNA is expressed strongly in the adult human kidney and weakly in the prostate, pituitary gland, lung, liver, thyroid gland, and small intestine. The mRNA was either at very low levels or not detectable in the fetal kidney, lung, and liver. Thus, expression of the human arginase II gene is regulated both tissue-specifically and developmentally.

Adult↗

PubMed1998

Arginase AI is upregulated in acute immune complex-induced inflammation.

Previous studies have shown high arginase activity at inflammatory sites. Arginase converts L-arginine to L-ornithine, sharing a common substrate with nitric oxide synthase. It exists as two isoforms, AI and AII. While the function of liver arginase (AI) in ureagenesis has been defined, the role and isoform of arginase in cells without a complete urea cycle are unknown. We therefore determined arginase isoform mRNA expression in glomerular acute immune complex inflammation, and its cultured constituent cells. AI was induced in nephritic glomeruli, and in mesangial cells stimulated with IL-4 and cAMP, and was present in elicited neutrophils and macrophages. AII was constitutively expressed. Our data strongly suggest that AI, thought to be restricted to the liver, accounts for high arginase activity at inflammatory sites where it may limit high output nitric oxide production and generate polyamines and proline essential for cell proliferation and matrix production. This identification of AI in inflamed tissue is an important step for understanding the consequences of increased arginase activity.

Acute Disease↗

PubMed2001

Increased expression of arginase II in human diabetic corpus cavernosum: in diabetic-associated erectile dysfunction.

Nitric oxide (NO) is the principal mediator of penile erection. NO is synthesized by nitric oxide synthase (NOS). It has been well documented that the major causative factor contributing to erectile dysfunction in diabetic patients is the reduction in the amount of NO synthesis in the corpora cavernosa of the penis resulting in alterations of normal penile homeostasis. Arginase is an enzyme that shares a common substrate with NOS, thus arginase may downregulate NO production by competing with NOS for this substrate, l-arginine. The purpose of the present study was to compare arginase gene expression, protein levels, and enzyme activity in diabetic human cavernosal tissue. When compared to normal human cavernosal tissue, diabetic corpus cavernosum from humans with erectile dysfunction had higher levels of arginase II protein, gene expression, and enzyme activity. In contrast, gene expression and protein levels of arginase I were not significantly different in diabetic cavernosal tissue when compared to control tissue. The reduced ability of diabetic tissue to convert l-arginine to l-citrulline via nitric oxide synthase was reversed by the selective inhibition of arginase by 2(S)-amino-6-boronohexanoic acid (ABH). These data suggest that the increased expression of arginase II in diabetic cavernosal tissue may contribute to the erectile dysfunction associated with this common disease process and may play a role in other manifestations of diabetic disease in which nitric oxide production is decreased.

Arginase↗

PubMed2002

Upregulation of arginase expression in wound-derived fibroblasts.

BACKGROUND: Wound-derived fibroblasts (WFBs) are phenotypically different from normal dermal fibroblasts (NFBs). We have previously shown that the wound phenotype correlates with expression of the inducible isoform of nitric oxide synthase (iNOS) in fibroblasts. l-Arginine is the sole substrate for iNOS. Arginase is an alternative pathway of l-arginine metabolism in wounds. To clarify the role of l-arginine in wound healing, we investigated arginase expression and activity in WFB. METHODS: Male Lewis rats underwent dorsal skin incisions and subcutaneous PVA sponge implantation. WFBs were harvested from sponges retrieved at different days postimplantation. Normal fibroblasts were obtained from uninjured skin by an explant technique. Arginase activity was measured by newly formed urea (nmol/min/mg protein) and protein expression was detected by Western blotting using specific antibodies for type I (AI) and type II (AII). The effect of transforming growth factor beta1 (TGF-beta1), interleukin-4 (IL-4), lipopolysaccharide, and wound fluid on arginase activity was also investigated. RESULTS: WFB arginase activity was significantly elevated compared with NFB activity at all times postwounding. This was paralleled by increased AI protein expression by Western blotting. AII was not detectable. TGF-beta and IL-4 significantly increased arginase activity and protein expression whereas lipopolysaccharide and wound fluid did not affect it. CONCLUSIONS: The upregulation of the arginase expression in WFB underlines the distinct regulation of l-arginine metabolism in WFBs. Further work is needed to elucidate the functional implications.

Adjuvants, Immunologic↗

PubMed2000

Phosphodiesterase inhibitors and forskolin Up-regulate arginase activity in rabbit alveolar macrophages.

Alveolar macrophages (AMsmall ef, Cyrillic) express considerable arginase activity which can be modulated by various mediators. As inhibitors of phosphodiesterase (PDE) play an increasing role in the treatment of chronic inflammatory and obstructive airway disease, we tested whether PDE inhibitors affect arginase activity in AMsmall ef, Cyrillic. Isolated rabbit AMsmall ef, Cyrillic were cultured for 20 h in the absence or presence of bacterial lipopolysaccharides (LPS) and/or different test substances. Thereafter arginase activity was determined by measuring the formation of [(3)H]-L-ornithine during 1 h incubation with [(3)H]-L-arginine. Lipopolysaccharide-enhanced (0. 01-5 microg/ml) maximal arginase activity by about 2.5-fold. The non-selective PDE inhibitor IBMX and the PDE4 selective inhibitor rolipram (each up to 30 microM) caused a 2.4-fold increase in arginase activity, and these effects were additive to those of LPS. The PDE3-selective inhibitor siguazodan had only marginal effects. Forskolin (10 microM) also enhanced arginase activity in the absence and presence of LPS. The effect of forskolin was almost prevented by cycloheximide (30 microM) and largely attenuated by the protein kinase A inhibitor KT 5720 (300 nM). In conclusion, inhibition of the cAMP-specific PDE4, like direct activation of adenylyl cyclase, causes an up-regulation of arginase activity in rabbit AMsmall ef, Cyrillic.

1-Methyl-3-isobutylxanthine↗

PubMed1999

Identification of two arginase isoenzyme activities along the nephron of Meriones shawi.

Conflicting theories on the existence of several renal arginase isoenzymes remain in debate. Because the activity of arginase is high in two embryologically different nephron segments of the Meriones shawi kidney, namely the cortical (CPST) and medullary (OSPST) proximal straight tubule and the outer medullary collecting duct (OMCD), we postulate that these nephron segments may contain different isoforms. Isolated nephron segments were dissected from collagenase-treated kidneys. Tubules were permeabilized with Triton X-100 (0.25%) and incubated with increasing Arg concentrations to characterize the arginase activity. The results were as follows: (1) in OMCD, one arginase isoform (E1), characterized by a high Arg affinity (1.160 mM), was present; (2) in CPST, two arginase isoforms were discovered - one, E1, had a similar Km (1.407 mM) to that found in OMCD whereas the other (E2) had a low affinity for Arg (Km =18.8 mM); and (3) in OSPST, two isoenzymes were present - E1 which had a Km of 1.478 mM and the second isoform that we named E2 which had a Km of 9.07 mM. In addition, arginase located in CPST and OMCD was strongly inhibited by Orn and Lys. The Ki value for Lys varied between 1.635 and 2.288 mM. Therefore, this work demonstrates that two arginase isoforms are present in the kidney of Meriones shawi. Isoform E1 is present in the proximal tubule and the collecting duct whereas isoform E2 is restricted to the proximal tubule.

Animals↗

PubMed2003

Loblolly pine arginase responds to arginine in vitro.

Following germination of loblolly pine (Pinus taeda L.) seeds, storage proteins in the embryo and megagametophyte are broken down to provide nitrogen, in the form of amino acids, to the developing seedling. A substantial portion of the free amino acids released in this process is arginine. Arginine is hydrolyzed in the cotyledons of the seedling by the enzyme arginase (EC 3.5.3.1), which is under developmental control. It has been shown previously that the seedling is able to initiate arginase gene expression in vitro in the absence of the megagametophyte, however, presence of the megagametophyte causes a greater accumulation of arginase protein and mRNA. Using an in vitro culture system we show that arginine itself may be responsible for up-regulating arginase activity. Application of exogenous arginine to cotyledons of seedlings germinated in the absence of the megagametophyte caused an increase in total shoot pole arginase activity as well as arginase specific activity. Arginine was also able to induce arginase mRNA accumulation in the same tissue.

Arginase↗

PubMed1981

Arginase activity in human fibroblast cultures.

Controversial results have been published as to the presence of arginase activity in human skin fibroblasts in normal cells and in argininemia. Experiments were undertaken to see if arginase is intrinsic to the fibroblasts or may be there as the result of exogenous contamination inherent to the mode of cell culturing. The mode of harvesting by scraping or trypsin treatment demonstrated similar arginase activity. No significant differences of arginase activity were found between the fibroblasts grown on fetal calf or human serum. In the conditioned serum-free medium arginase separated on DEAE-cellulose into an A1 and A4 form. These fractions are identical with those found in the cells. Arginase activity varies, however, with age of the fibroblasts. These observations are also valid for fibroblasts from a case of argininemia. Arginase activity is therefore intrinsic to diploid human fibroblasts.

Amino Acid Metabolism, Inborn Errors↗

PubMed1983

Arginase activity of different cells in tissue culture.

Arginase activity was measured in ten different cell lines and all of them showed arginase activity. However, the amount of the activity in the cells varied more than 1000-fold. The cell density did not appear to affect the arginase activity much, since cultures of human fibroblasts grown to different cell densities exhibited only small variations in arginase activity. Arginase activity was in general higher than that of ornithine decarboxylase and there was no correlation between the two activities. When human fibroblasts were stimulated to proliferate in serum-free medium by adding certain growth factors the activity of ornithine decarboxylase increased markedly prior to the DNA synthesis, while the arginase activity increased more slowly and to a much smaller degree. Two cell lines, baby hamster kidney and mouse 3T3 cells, had low arginase activity when adapted to grow in serum-free medium, but in spite of this they were able to grow in serum-free medium without exogenous ornithine.

Animals↗

PubMed1992

Arginase distribution in tissues of domestic animals.

1. A new colorimetric method was used for determination of arginase in different tissues of some domestic animals. 2. In all species studied liver was the richest source of arginase. 3. Significant differences were observed in the specific activity of arginase in livers from different species. 4. In all species, besides liver, kidney and brain also contained significant levels of arginase. 5. In the dog, in addition to the three organs mentioned above, lung, heart, spleen and skeletal muscle showed some arginase activity. 6. In sheep and cattle significant arginase activity was observed in the rumen. No differences were observed between epithelial and muscular layers of different parts of digestive system in all species studied. 7. These results are discussed in terms of the possible role of arginase in different tissues of animals.

Animals↗

PubMed1988

The isolation and immunological properties of two arginase forms from human erythrocytes.

1. Two forms of arginase were isolated from human erythrocytes; the main form adsorbed on CM-cellulose and the second form, occurring in much smaller amount, adsorbed on DEAE-cellulose. 2. The molecular weight of either arginase was 120,000 +/- 5000. 3. The erythrocyte arginases are similar in immunological properties to arginase A4 from human kidney and A2 from human liver, respectively. 4. Despite the literature data stating that human erythrocyte arginase and human liver arginase are identical, it was found that the main forms of arginase of these tissues A4 from erythrocytes and A5 from liver differ in immunological properties.

Arginase↗

PubMed2004

The ornithine cycle enzyme arginase from Agaricus bisporus and its role in urea accumulation in fruit bodies.

An extensive survey of higher fungi revealed that members of the family Agaricaceae, including Agaricus bisporus, accumulate substantial amounts of urea in their fruit bodies. An important role of the ornithine cycle enzymes in urea accumulation has been proposed. In this work, we present the cloning and sequencing of the arginase gene and its promoter region from A. bisporus. A PCR-probe based on fungal arginase was used to identify the A. bisporus arginase gene from a cDNA library. The arginase cDNA encodes a 311-aa protein which is most likely expressed in the cytosol. Expression of the cDNA in Escherichia coli was established as a His-tagged fusion protein. The arginase gene was used as a molecular marker to study expression and regulation during sporophore formation and postharvest development. The expression of the arginase gene was significantly up-regulated from developmental stage 3 onwards for all the tissues studied. A maximum of expression was reached at stage 6 for both stipe and cap tissue. In postharvest stages 5, 6 and 7 the level of expression observed was similar to normal growth stages 5, 6 and 7. A good correlation was found between arginase expression and urea content of stipe, velum, gills, cap and peel tissue. For all tissues the urea content decreased over the first four stages of development. From stage 4 onwards urea accumulated again except for stipe tissue where no significant changes were observed. The same trend was also observed for postharvest development, but the observed increase of urea in postharvest tissues was much higher.

Agaricus↗

PubMed2006

Arginase activity is increased by thrombin: a mechanism for endothelial dysfunction in arterial thrombosis.

BACKGROUND: Earlier observations implicate arterial thrombosis causing endothelial dysfunction by decreasing nitric oxide (NO) levels. NO levels are restored by regional L-arginine supplementation in animal models. The purpose of this study was to investigate the roles of thrombus components in NO generation. STUDY DESIGN: Human umbilical vein endothelial cells were harvested and cultured. The thrombus components thrombin, thrombin receptor agonist peptide (TRAP), and fibrin were added to a media of confluent human umbilical vein endothelial cells. Endothelial nitric oxide synthase (eNOS) activity was assayed by measuring conversion of L-arginine to L-citrulline. Endothelial NOS mRNA levels were quantitated using real-time polymerase chain reaction. Cellular membrane transport of L-arginine through the y+ channel was assayed with (14)C-labeled L-arginine. Arginase activity was determined as the conversion of (14)C L-arginine to (14)C urea and trapped as Na(2)(14)CO(3) for scintillation counting. Arginase protein amounts were assessed using Western blotting. RESULTS: Endothelial cells exposed to thrombin for 4 hours led to increased arginase activity. Thrombin (10 U/mL) caused a 1.6-fold increase compared with that in controls (320+/-29 microM urea/min versus 194+/-10 microM urea/min, p=0.03), and thrombin (30 U/mL) increased arginase activity 2.1-fold (398+/-27 microM urea/min, p < 0.001, versus controls); thrombin at 1 U/mL and fibrin had no effect. TRAP (50 microM) had an effect similar to that of thrombin 10 U/mL (316+/-21 microM urea/min, p < 0.01, versus controls). Protein amounts of arginase corresponded with activity levels. Neither eNOS nor inducible nitric oxide synthase (iNOS) activities were affected by exposure to thrombin and TRAP for 4 hours. Similarly, quantification of eNOS, iNOS, and endothelin-1 mRNA did not change, although CL-100, a known thrombin-inducible gene, was upregulated. Finally, transport of L-arginine into endothelial cells was unaffected by thrombin, TRAP, and fibrin exposure. CONCLUSIONS: Endothelial cells exposed to thrombin have increased arginase enzymatic activity, and the remainder of NO generation capability is unaffected. L-arginine supplementation or arginase blockade may counteract endothelial dysfunction in the setting of acute arterial thrombosis.

Arginase↗

PubMed2004

Hyperargininemia due to liver arginase deficiency.

The urea cycle is a series of six reactions necessary to rid the body of the nitrogen generated by the metabolism, primarily of amino acids, from the diet or released as the result of endogenous protein catabolism. Arginase is the sixth and final enzyme of this cycle. Arginase catalyzes the conversion of arginine to urea and ornithine, the latter recycled to continue the cycle. Hyperargininemia due to arginase deficiency is inherited in an autosomal recessive manner and gene for arginase, designated AI, has been cloned. Unlike the other urea cycle enzymes, a second gene encoding arginase, with similar structural properties and enzyme characteristics, exists and has been named Arginase II (AII). Comprehensive histories and physical examinations confirm a strikingly uniform clinical picture and one notably different from patients with other urea cycle disorders. This condition rarely presents in the neonatal period and first symptoms typically present in children between 2 and 4 years of age. First symptoms are often neurologically based. If untreated, symptoms are progressive with a gradual loss of developmental milestones. With adherence to a dietary and drug regimen, a favorable outcome can be expected, with cessation of further neurological deterioration and in some instances, of improvement. This article summarizes the clinical course of selected patients who represent the full spectrum of presentations of arginase deficiency. In addition to the clinical characterization of this disorder; the biochemical, enzymatic, and molecular evidence of disease is summarized. Treatment and prenatal diagnosis are also discussed.

Arginase↗

PubMed2003

Arginase in patients with breast cancer.

The mean arginase activity in breast cancers (n = 80) was significantly higher than in control tissues and it accounted for 0.31 +/- 0.23 U/g wet tissue and 0.083 +/- 0.061 U/g (P < 0.05), respectively. With the cutoff value of 0.1 U/g wet tissue, raised arginase activity was observed in 74% of tumors. The preoperative arginase activity in blood serum from women with breast cancer was 11.2 +/- 7.9 U/l (n = 115), and it was significantly higher than in 70 healthy controls, where it was 5.7 +/- 2.4 U/l (P < 0.05). With the cutoff value for normal serum arginase activity above 8.0 U/l, the activity was raised in 10% of control individuals, and in 63% of women with breast cancer. The sensitivity and specificity of the arginase test in blood serum were 63% and 60%, respectively. Two isoforms immunologically identical to human kidney arginases (L-arginine amidinohydrolase) were found in both normal and cancerous breast tissues. The level of anionic form was similar in control and cancerous tissues, whereas the cationic isoform predominated in breast cancer. The cationic isoform was the only one present in serum of both ill and healthy women, and its level was higher in patients with breast cancer. Thus, it can be concluded that the cationic isoform is responsible for the increase of arginase activity in serum of patients with breast cancer.

Adult↗

PubMed1998

Liver-type arginase in serum during and after liver transplantation: a novel index in monitoring conditions of the liver graft and its clinical significance.

We quantified liver-type arginase in sera of 47 patients undergoing partial liver transplantation with use of an ELISA method. The level of liver-type arginase fluctuated slightly beyond the normal range in successful liver recipients, while it changed more drastically or precipitously in unsuccessful ones, accompanying or unaccompanying elevation of AST and ALT levels. A higher elevation pattern of the arginase level (above 100 ng ml-1) was observed in each of the unsuccessful recipients with critical condition, except for one patient. Other hepatic markers (LDH, ALP, and T-BIL) remained relatively unchanged until the terminal stage of deceasing patients. The finding that the liver-type arginase emerged in large quantity in the blood stream immediately after reperfusion of the liver graft indicates that the enzyme leaks out of hepatocytes damaged, presumably, by storage in the absence of circulation. A half-life of the liver-type arginase in the human blood was estimated to be 1 h, that is clearly shorter than that of AST. The short half-life of the arginase appears to be ascribable, at least partly, to formation of an immune complex with circulating autoantibody which appears in many liver recipients. These results suggest that liver-type arginase behaves uniquely in the serum among many hepatic enzymes, and could serve as a distinct marker of hepatic lesions, particularly during and after liver transplantation.

Adolescent↗

PubMed2002

Arginase expression and modulation of IL-1beta-induced nitric oxide generation in rat and human islets of Langerhans.

Proinflammatory cytokine induction of NO synthesis may contribute to the destruction of pancreatic beta cells leading to type 1 diabetes. The NO synthase substrate arginine can also be metabolized to ornithine and urea in a reaction catalyzed by cytosolic (AI) or mitochondrial (AII) isoforms of arginase. Recent evidence suggests that the rate of NO generation is dependent on the relative activities of NO synthase and arginase. The objectives of this study were (i). to identify the arginase isoforms expressed in rat and human islets of Langerhans and a rat beta cell line, RINm5F and (ii). to investigate the competition for arginine between NO synthase and arginase in IL-1beta-treated rat islets. Arginase activity was detected in rat islets (fresh tissue, 346 mU/mg protein; cultured, 587 mU/mg), cultured human islets (56 mU/mg), RINm5F cells (376 mU/mg), rat kidney (238 mU/mg), and rat liver (6119 mU/mg). Using Western blots, AI was shown to be the predominant isoform expressed in rat islets and in RINm5F cells while human islets expressed far more AII than AI. Rat islets were cultured in medium containing 1.14, 0.1, and 0.01 mM arginine and treated with IL-1beta and the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH). IL-1beta-induced NO generation was unaffected by ABH at 1.14 mM arginine, but significantly increased at 0.1 and 0.01 mM arginine. These findings suggest that the level of islet arginase activity can regulate the rate of induced NO generation and this may be relevant to the insulitis process leading to beta cell destruction in type 1 diabetes.

Aminocaproates↗

PubMed2001

Probing erectile function: S-(2-boronoethyl)-L-cysteine binds to arginase as a transition state analogue and enhances smooth muscle relaxation in human penile corpus cavernosum.

The boronic acid-based arginine analogue S-(2-boronoethyl)-L-cysteine (BEC) has been synthesized and assayed as a slow-binding competitive inhibitor of the binuclear manganese metalloenzyme arginase. Kinetic measurements indicate a K(I) value of 0.4-0.6 microM, which is in reasonable agreement with the dissociation constant of 2.22 microM measured by isothermal titration calorimetry. The X-ray crystal structure of the arginase-BEC complex has been determined at 2.3 A resolution from crystals perfectly twinned by hemihedry. The structure of the complex reveals that the boronic acid moiety undergoes nucleophilic attack by metal-bridging hydroxide ion to yield a tetrahedral boronate anion that bridges the binuclear manganese cluster, thereby mimicking the tetrahedral intermediate (and its flanking transition states) in the arginine hydrolysis reaction. Accordingly, the binding mode of BEC is consistent with the structure-based mechanism proposed for arginase as outlined in Cox et al. [Cox, J. D., Cama, E., Colleluori D. M., Pethe, S., Boucher, J. S., Mansuy, D., Ash, D. E., and Christianson, D. W. (2001) Biochemistry 40, 2689-2701.]. Since BEC does not inhibit nitric oxide synthase, BEC serves as a valuable reagent to probe the physiological relationship between arginase and nitric oxide (NO) synthase in regulating the NO-dependent smooth muscle relaxation in human penile corpus cavernosum tissue that is required for erection. Consequently, we demonstrate that arginase is present in human penile corpus cavernosum tissue, and that the arginase inhibitor BEC causes significant enhancement of NO-dependent smooth muscle relaxation in this tissue. Therefore, human penile arginase is a potential target for the treatment of sexual dysfunction in the male.

Animals↗