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^ω-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.     

Arginase activity in the frog urinary bladder epithelial cells and its involvement in regulation of nitric oxide production

The activity of arginase converting arginine into ornithine and urea is of particular interest among many factors regulating NO production in the cells. It is known that by competing with NO-synthase for common substrate, arginase can affect the NO synthesis. In the present work, the properties of arginase from the frog Rana temporaria L. urinary bladder epithelial cells possessing the NO-synthase activity were characterized, and possible contribution of arginase to regulation of NO production by epithelial cells was studied. It has been shown that the enzyme had the temperature optimum in the range of 55-60 degrees C, K(m) for arginine 23 mM, and V(max) about 10 nmol urea/mg protein/min, and its activity was effictively inhibited by (S)-(2-boronoethyl)-L-cysteine (BEC), an inhibitor of arginase, at concentrations from 10(-6) to 10(-4) M. The comparison of arginase activity in various frog tissues revealed the following pattern: liver > kidney > brain > urinary bladder (epithelium) > heart > testis. The arginase activity in the isolated urinary bladder epithelial cells was 3 times higher than that in the intact urinary bladder. To evaluate the role of arginase in the regulation of NO production, epithelial cells were cultivated in the media L-15 or 199 containing different amounts of arginine; the concentration of NO2-, the stable NO metabolite, was determined in the culture fluid after 18-20 h of cells incubation. The vast majority of the produced nitrites are associated with the NOS activity, as L-NAME, the NOS-inhibitor, decreased their accumulation by 77.1% in the L-15 medium and by 80% in 199 medium. BEC (10(-4) M) increased the nitrite production by 18.0 % +/- 2.7 in the L-15 medium and by 24.2 +/- 3.5 in the 199 medium (p < 0.05). The obtained data indicate a relatively high arginase activity in the frog urinary bladder epithelium and its involvement in regulation of NO production by epithelial cells.     

Changes in arginase isoenzymes pattern in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide affecting preferentially patients with liver cirrhosis. The studies were performed on tissues obtained during surgery from 50 patients with HCC, 40 with liver cirrhosis and 40 control livers. It was found that arginase activity in HCC was nearly 5- and 15-fold lower than in cirrhotic and normal livers, respectively. Isoenzymes AI (so-called liver-type arginase) and AII (extrahepatic arginase) were identified by Western blotting in all studied tissues, however the amount of AI, as well as the expression of AI-mRNA were lower in HCC, in comparison with normal liver, and those of AII were significantly higher. Since HCC is arginine-dependent, and arginine is essential for cells growth, the decrease of AI may preserve this amino acid within tumor cells. Concurrently, the rise of AII can increase the level of polyamines, compounds crucial for cells proliferation. Thus, both arginase isoenzymes seem to participate in liver cancerogenesis.     

Ornithine cycle in Nostoc PCC 73102. Arginase,OCT and arginine deiminase,and the effects of addition of external arginine,ornithine, or citrulline

Arginase, ornithine carbamoyl transferase (OCT) and arginine deiminase activities were found in cell-free extracts of Nostoc PCC 73102, a free-living cyanobacterium originally isolated from the cycad Macrozamia. Addition of either arginine, ornithine or citrulline to the growth medium induced significant changes in their in vitro activities. Moreover, growth in darkness, compared to in light, induced higher in vitro activities. The in vitro activities of arginase and arginine deiminase, two catabolic enzymes primarily involved in the breakdown of arginine, increased substantially by a combination of growth in darkness and addition of either arginine, or ornithine, to the growth medium. The most significant effects on the in vitro OCT activities where observed in cells grown with the addition of ornithine. Cells grown in darkness exhibited about 6% of the in vivo nitrogenase activity observed in cells grown in light. However, addition of external carbon (glucose and fructose) to cells grown in darkness resulted in in vivo nitrogenase activity levels similar to, or even higher than, cells grown in light. Growth with high in vivo nitrogenase activity or in darkness with the addition of external carbon, resulted in repressed levels of in vitro arginase and arginine deiminase activities. It is suggested that nitrogen starvation induces a mobilization of the stored nitrogen, internal release of the amino compound arginine, and an induction of two catabolic enzymes arginase and arginine deiminase. A similar and even more pronunced induction can be observed by addition of external arginine to the growth medium.     

Diclofenac Inhibits Tumor Growth in a Murine Model of Pancreatic Cancer by Modulation of VEGF Levels and Arginase Activity

Background

Diclofenac is one of the oldest anti-inflammatory drugs in use. In addition to its inhibition of cyclooxygenases (COX), diclofenac potently inhibits phospholipase A₂ (PLA₂), thus yielding a broad anti-inflammatory effect. Since inflammation is an important factor in the development of pancreatic tumors we explored the potential of diclofenac to inhibit tumor growth in mice inoculated with PANCO2 cells orthotopically.

Methodology/Principal Findings

We found that diclofenac treatment (30 mg/kg/bw for 11 days) of mice inoculated with PANC02 cells, reduced the tumor weight by 60%, correlating with increased apoptosis of tumor cells. Since this effect was not observed in vitro on cultured PANCO2 cells, we theorized that diclofenac beneficial treatment involved other mediators present in vivo. Indeed, diclofenac drastically decreased tumor vascularization by downregulating VEGF in the tumor and in abdominal cavity fluid. Furthermore, diclofenac directly inhibited vascular sprouting ex vivo. Surprisingly, in contrast to other COX-2 inhibitors, diclofenac increased arginase activity/arginase 1 protein content in tumor stroma cells, peritoneal macrophages and white blood cells by 2.4, 4.8 and 2 fold, respectively. We propose that the subsequent arginine depletion and decrease in NO levels, both in serum and peritoneal cavity, adds to tumor growth inhibition by malnourishment and poor vasculature development.

Conclusion/Significance

In conclusion, diclofenac shows pronounced antitumoral properties in pancreatic cancer model that can contribute to further treatment development. The ability of diclofenac to induce arginase activity in tumor stroma, peritoneal macrophages and white blood cells provides a tool to study a controversial issue of pro-and antitumoral effects of arginine depletion.     

Hyperargininemia: the rat as a model for the human disease and the comparative response to enzyme replacement therapy with free arginase and arginase-loaded erythrocytes in vivo

Rat erythrocytes lack arginase as do the erythrocytes of human homozygote patients with hyperargininemia due to arginase deficiency. The rat has physiological liver arginase activity and plasma arginine and ornithine levels between the homozygotes and the heterozygotes with hyperargininemia. In rats, one injection of free arginase induces a transient exogenous arginase effect which is abolished after 24 hr. One injection of isoionic arginase-loaded erythrocytes provokes an exogenous arginase effect in physiological "hyperargininemic" rats and pathological "hyperargininemic"-made rats for at least 8 and 5 days respectively. The very transient response in vivo to exogenous free arginase can be considerably prolonged by entrapment of the arginase in isoionic prepared erythrocytes.     

Functional heterogeneity among peritoneal macrophages: III. No evidence for the role of arginase in the inhibition of tumor cell growth by supernatants from macrophages or macrophage subpopulation cultures

The adherent population of peritoneal exudate cells (PE) obtained from rats and mice was analyzed for arginase activity in order to determine whether this enzyme has a role in tumor-growth-inhibitory activity. Freshly obtained tumor-growth-inhibitory rat PE cells had little or no arginase activity compared to the high levels of enzyme activity of mouse PE cells. Even after culturing, rat PE cells contained arginase activity 10 times less than that observed with comparable numbers of cultured or noncultured mouse cells. Subpopulations of mouse and rat PE macrophages, analyzed for arginase activity, showed that the light-density populations from cultured rat PE cells and noncultured mouse PE cells expressed arginase activities greater than that seen with heavy-density cells. However, the light-density rat PE cells expressed significantly less arginase activity than did the mouse cells. In attempts to test whether the inability of tumor cells to grow in supernatants or dialyzed supernatants from PE macrophage cultures is due to an arginine depletion, 200 μg/ml of the amino acid was added to the supernatants. The tumor-growth-inhibitory activities of such supernatants, as well as those from supernatants from highly active light-density rat PE macrophage cultures, were not abrogated by the addition of arginine. There was no correlation between the high levels of arginase activity of light-density PE macrophages and their antitumor activity and no evidence that the tumor-growth-inhibitory activity of rat or mouse PE macrophages in the macrophage-tumor models we studied was due to an arginine depletion.     

Arginase as one of the inhibitory principles in the density-dependent as well as plasma membrane-mediated inhibition of liver cell growth in vitro

Liver cells isolated from the adult rat livers under mild conditions were preincubated for 1 day with Williams medium E (WE) containing serum, dexamethasone and insulin, and then the cells (monolayered) were incubated for 2-3 days with WE (1 ml) containing only insulin to measure DNA synthesis and/or mitosis. DNA synthesis of cultured liver cells was dependent on cell densities within a region from 0.1 X 10(6) to 1.0 X 10(6) nuclei/dish (Falcon, diameter 35 mm). The addition of EGF from the beginning of preincubation stimulated DNA synthesis (or replication) as well as cell proliferation in vitro, but the density-dependent inhibition of DNA synthesis was observed similarly in the presence of EGF. In contrast to the low and high density cultures, DNA synthesis in the intermediary density cultures was enhanced by enlarging the medium volume or by adding ornithine (arginase inhibitor). DNA synthesis in low density cultures was inhibited by liver plasma membranes in a concentration-dependent fashion. The inhibition of DNA synthesis by liver plasma membranes in low concentrations (less than 30 micrograms protein/ml) was reduced by adding either extra arginine or ornithine. DNA synthesis of cultured liver cells (low density) was inhibited by replacing arginine in WE with equimolar ornithine and urea or by adding a commercial arginase (bovine liver). These, together with earlier findings indicating the presence of arginase in liver plasma membranes (outer leaflet), seem to support the idea that arginase may be involved in density-dependent as well as plasma membrane-mediated inhibition of DNA synthesis of cultured liver cells. However, this does not exclude possible involvement of other inhibitory principle(s), such as direct cell-to-cell or cell-to-plasma membrane interactions, especially in higher cell densities or larger plasma membrane concentrations.     

Nitrogen regulation of arginase in Neurospora crassa.

The final products of the arginine catabolism that can be utilized as a nitrogen source in Neurospora crassa are ammonium, glutamic acid, and glutamine. The effect of these compounds on arginase induction by arginine was studied. In wild-type strain 74-A, induction by arginine was almost completely repressed by glutamic acid plus ammonium, whereas ammonium or glutamic acid alone had only moderate effects. Arginine products of catabolism also repressed arginase induction. A mutant, ure-1, which lacks urease activity, hyperinduced its arginase with arginine as a nitrogen source. The addition of either ammonium or glutamine produced effects similar to those in the wild-type strain. The effect of ammonium on arginase induction is mediated through its conversion into glutamine. This was demonstrated in mutant am-1, which lacks L-glutamate dehydrogenase activity. In this mutant, the effect of glutamic acid was reduced, and, with ammonium, it was completely lost. The addition of glutamine or glutamic acid plus ammonium to this strain decreased by threefold the induction of arginase by arginine. Proline, a final product of arginine catabolism, competitively inhibited arginase activity. This effect and the repression of arginase by glutamine are examples of negative modulation of the first enzyme in a catabolic pathway by its final products.     

Arginase activity in frog urinary bladder epithelial cells and its involvement in regulation of nitric oxide production

The activity of arginase converting arginine into ornithine and urea is of particular interest among many factors regulating NO production in the cells. It is known that by competing with NO-synthase for common substrate (arginine), arginase can affect NO synthesis. In the present work, properties of arginase from the common frog Rana temporaria L. urinary bladder epithelial cells containing the NO-synthase were characterized, and possible contribution of arginase to regulation of NO production by epithelial cells was studied. It has been shown that the enzyme has temperature optimum in the range of 55–60°C, K _M for arginine 23 mM, and V _max about 10 nmole urea/mg of protein/min, and its activity was efficiently inhibited by (S)-(2-boronoethyl)-L-cysteine (BEC), an inhibitor of arginase, at concentrations from 10^?6 to 10^?4 M. The comparison of arginase activity in various frog tissues revealed the following pattern: liver > kidney > brain > urinary bladder (epithelium) > heart > testis. The arginase activity in isolated urinary bladder epithelial cells was 3 times higher that in the intact urinary bladder wall. To evaluate the role of arginase in regulation of NO production, the epithelial cells were cultivated in the media L-15 or 199 containing different amounts of arginine; the concentration of NO₂ ^?, the stable NO metabolites, was de-termined in the cultural fluid after 18–20 h of cell incubation. The vast majority of the produced nitrites are associated with NOS activity, as L-NAME, the NO inhibitor, decreased their accumulation by 77.1% in the L-15 medium and by 80% in the 199 medium. BEC (10^?4 M) increased nitrite production by 18.0% ± 2.7% in the L-15 medium and by 24.4% ± 3.5% in the 199 medium (p < 0.05). The obtained data indicate a relatively high activity of arginase in the frog urinary bladder epithelium and its involvement in regulation of NO production.     

Production and characterization of monoclonal antibodies to rat liver arginase

Rat liver arginase was purified and five monoclonal antibodies were produced by fusion of spleen cells from a Balb/c mouse and the myeloma cell line P3-X36-Ag-U1. One, R2D19, of five antibodies belonged to the IgG2a subclass, the other four, R1D81, R1G11, R2E10, and R2G51, were of the IgG1 type. The R1D81 cross-reacted with human liver arginase. This antibody inhibited the arginase activity, competing with arginine. These results suggest that R1D81 binds to the catalytic site of arginase. The R2D19 also inhibited the enzyme activity but acted as a noncompetitive inhibitor. With the use of R1D81 and a polyclonal anti-human liver arginase antibody conjugated with alkaline phosphatase, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of human arginase. Specificity of monoclonal antibodies for rat liver arginase was examined by means of the sandwich ELISA. Eight pairs of monoclonal antibodies could form a sandwich with the arginase. Only the R2E10 could be used for both the first and the second antibody in the sandwich system. In other cases, monoclonal antibodies could not be interchanged between solid and liquid phase.     

Liver I/R injury is improved by the arginase inhibitor, N(omega)-hydroxy-nor-L-arginine (nor-NOHA)

Liver ischemia-reperfusion (I/R) injury is associated with profound arginine depletion due to arginase release from injured hepatocytes. The purpose of this study was to determine whether arginase inhibition with N(omega)-hydroxy-nor-l-arginine (nor-NOHA) would increase circulating arginine levels and decrease hepatic damage during liver I/R injury. The effects of nor-NOHA were initially tested in normal animals to determine in vivo toxicity. In the second series of experiments, orthotopic syngeneic liver transplantation (OLT) was performed after 18 h of cold ischemia time in Lewis rats. Animals were given nor-NOHA (100 mg/kg) or saline before and after graft reperfusion. In normal animals treated with nor-NOHA, there were no histopathological changes to organs, liver enzymes, serum creatinine, or body weight. In the OLT model, animals treated with saline exhibited markedly elevated serum transaminases and circulating arginase protein levels. Nor-NOHA administration blunted the increase in serum arginase activity by 80% and preserved serum arginine levels at 3 h after OLT. Nor-NOHA treatment reduced post-OLT serum liver enzyme release by 50%. Liver histology (degree of necrosis) in nor-NOHA-treated animals was markedly improved compared with the saline-treated group. Furthermore, use of the arginase inhibitor nor-NOHA did not influence polyamine synthesis owing to the decrease in ornithine levels. Arginase blockade represents a potentially novel strategy to combat hepatic I/R injury associated with liver transplantation.     

Arginase activities and global arginine bioavailability in wild-type and ApoE-deficient mice: responses to high fat and high cholesterol diets

Increased catabolism of arginine by arginase is increasingly viewed as an important pathophysiological factor in cardiovascular disease, including atherosclerosis induced by high cholesterol diets. Whereas previous studies have focused primarily on effects of high cholesterol diets on arginase expression and arginine metabolism in specific blood vessels, there is no information regarding the impact of lipid diets on arginase activity or arginine bioavailability at a systemic level. We, therefore, evaluated the effects of high fat (HF) and high fat-high cholesterol (HC) diets on arginase activity in plasma and tissues and on global arginine bioavailability (defined as the ratio of plasma arginine to ornithine + citrulline) in apoE(-/-) and wild-type C57BL/6J mice. HC and HF diets led to reduced global arginine bioavailability in both strains. The HC diet resulted in significantly elevated plasma arginase in both strains, but the HF diet increased plasma arginase only in apoE(-/-) mice. Elevated plasma arginase activity correlated closely with increased alanine aminotransferase levels, indicating that liver damage was primarily responsible for elevated plasma arginase. The HC diet, which promotes atherogenesis, also resulted in increased arginase activity and expression of the type II isozyme of arginase in multiple tissues of apoE(-/-) mice only. These results raise the possibility that systemic changes in arginase activity and global arginine bioavailability may be contributing factors in the initiation and/or progression of cardiovascular disease.     

Arginase inhibition restores in vivo coronary microvascular function in type 2 diabetic rats

Nitric oxide (NO) is crucial for maintaining normal endothelial function and vascular integrity. Increased arginase activity in diabetes might compete with NO synthase (NOS) for their common substrate arginine, resulting in diminished production of NO. The aim of this study was to evaluate coronary microvascular function in type 2 diabetic Goto-Kakizaki (GK) rats using in vivo coronary flow velocity reserve (CFVR) and the effect of arginase inhibition to restore vascular function. Different groups of GK and Wistar rats were given vehicle, the arginase inhibitor N(ω)-hydroxy-nor-l-arginine (nor-NOHA), l-arginine, and the NOS inhibitor N(G)-monomethyl -l-arginine (l-NMMA). GK rats had impaired CFVR compared with Wistar rats (1.31 ± 0.09 vs. 1.87 ± 0.05, P < 0.001). CFVR was restored by nor-NOHA treatment compared with vehicle in GK rats (1.71 ± 0.13 vs. 1.23 ± 0.12, P < 0.05) but remained unchanged in Wistar rats (1.88 ± 0.10 vs. 1.79 ± 0.16). The beneficial effect of nor-NOHA in GK rats was abolished after NOS inhibition. CFVR was not affected by arginine compared with vehicle. Arginase II expression was increased in the aorta and myocardium from GK rats compared with Wistar rats. Citrulline-to-ornithine and citrulline-to-arginine ratios measured in plasma increased significantly more in GK rats than in Wistar rats after nor-NOHA treatment, suggesting a shift of arginine utilization from arginase to NOS. In conclusion, coronary artery microvascular function is impaired in the type 2 diabetic GK rat. Treatment with nor-NOHA restores the microvascular function by a mechanism related to increased utilization of arginine by NOS and increased NO availability.     

Identification of the apparently lymphocyte-specific human liver-derived inhibitory protein (LIP) as cytoplasmic liver L-arginase

This paper describes the identification of a human liver-derived inhibitory protein (LIP), which has recently been purified, as cytoplasmic liver arginase. Arginase activity was purified to homogeneity parallel to lymphocyte proliferation inhibitory activity. The reaction products were identified by thin-layer chromatography to be ornithine and urea from arginine. The enzyme activity could be increased by the addition of manganese ions, and the inhibitory effect on cell proliferation could be reversed by additional arginine. An antiserum against LIP cross-reacted with cytoplasmic calf liver arginase.     

Rat colon ornithine and arginine metabolism: coordinated effects after proliferative stimuli

Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.     

Purification and properties of arginase of rat kidney

l-Arginase from rat kidney was partially purified and some properties were compared with those of l-arginase of rat liver. The kidney enzyme was firmly bound to the mitochondrial fraction and after solubilization required arginine or an unknown factor in tissue extracts for stabilization after dialysis. The two enzymes differed also in stability with respect to acetone treatment, heating or freezing. In further contrast with liver arginase, arginase from kidney was not adsorbed to CM-cellulose at pH7.5 and its activity was not increased by incubation with Mn(2+). Other differences were seen in relative specificities for substrates, ratio of hydrolysis rates with high and low concentrations of arginine and effects of certain inhibitors. Antisera prepared to pure liver arginase did not cross-react with partially purified kidney arginase.     

Arginase II inhibited lipopolysaccharide-induced cell death by regulation of iNOS and Bcl-2 family proteins in macrophages

Arginase II catalyzes the conversion of arginine to urea and ornithine in many extrahepatic tissues. We investigated the protective role of arginase II on lipopolysaccharide-mediated apoptosis in the macrophage cells. Adenoviral gene transfer of full length of arginase II was performed in the murine macrophage cell line RAW264.7. The role of arginase II was investigated with cell viability, cytoplasmic histone-associated DNA fragmentation assay, arginase activity, nitric oxide production, and Western blot analysis. Arginase II is localized in mitochondria of macrophage cells, and the expression of arginase II was increased by lipopolysaccharide (LPS). LPS significantly increased cell death which was inhibited by AMT, a specific inducible nitric oxide synthase (iNOS) inhibitor. In contrast, LPS-induced cell death and nitric oxide production were increased by 2-boronoethyl-L-cysteine, a specific inhibitor of arginase. Adenoviral overexpression of arginase II significantly inhibited LPS-induced cell death and cytoplasmic histone-associated DNA fragmentation. LPS-induced iNOS expression and poly ADP-ribose polymerase cleavage were significantly suppressed by arginase II overexpression. Furthermore, arginase II overexpression resulted in a decrease in the Bax protein level and the reverse induction of Bcl-2 protein. Our data demonstrated that inhibition of NO production by arginase II may be due to arginine depletion as well as iNOS suppression though its reaction products. Moreover, arginase II plays a protective role of LPS-induced apoptosis in RAW264.7 cells.     

Control of a futile urea cycle by arginine feedback inhibition of ornithine carbamoyltransferase in Agrobacterium tumefaciens and Rhizobia

In Agrobacterium tumefaciens and Rhizobia arginine can be used as the sole nitrogenous nutrient via degradation by an inducible arginase. These microorganisms were found to exhibit arginine inhibition of ornithine carbamoyltransferase activity. This inhibition is competitive with respect to ornithine (Km for ornithine = 0.8 mM; Ki for arginine = 0.05 mM). This type of urea cycle regulation has not been observed among other microorganisms which degrade arginine via an arginase. The competitive pattern of this inhibition leads to its being inoperative in ornithine-grown cells, where the intracellular concentration of ornithine is high. In arginine-grown cells, however, the intracellular arginine and ornithine concentrations are compatible with inhibition and ornithine recycling appears to be effectively blocked in vivo.