Arginase activity patterns and their regulation during embryonic development in liver and kidney

The developmental patterns for mouse liver and kidney arginase were measured by a sensitive radioactive assay from day 8 of gestation until adulthood. On day 8 high arginase activity is generally distributed throughout early embryos. Then, as development proceeds, the arginase activity drops rapidly in liver and kidney, apparently because of mass increase unaccompanied by net arginase synthesis. Suddenly, on day 12 of gestation in liver and on day 16 in kidney, arginase activity begins to accelerate toward adult values.In order to study the mechanisms controlling arginase acceleration, 12- and 13-day fetal livers were explanted to organ cultures containing various exogenous chemicals, and subsequently assayed for arginase. Physiological concentrations of hydrocortisone causes the arginase activity to rise more than 100-fold to adult levels within 4 days in culture. Glucagon, thyroxine, and dibutyryl adenosine-3′-5′-cyclic phosphate have no effect in this system. Experiments with cycloheximide, actinomycin D, and 5-fluorodeoxyuridine suggest that the hydrocortisone response is dependent upon protein and RNA synthesis but independent of DNA synthesis.     

Studies on L-arginase in developing rat small intestine, brain, and kidney. I. Ontogenic evolution of arginase isoenzymes

The adult patterns of arginase isoenzymes in rat intestine, kidney, and brain are nearly identical and consist of two forms, cationic A1 and anionic A4. In this paper, the organ-specific maturation of the enzyme equipment in these tissues is reported. The activity of arginase in all tissues studied could be detected on the 13th to 16th days of gestation. In fetal intestine and kidney the arginase activity is low, and persists up to the weaning time when the rapid, 10-fold rise of the enzyme activity occurs. However, the adult pattern of arginase isoenzymes in these tissues is accomplished in different ways. In the intestine, arginase A1 appears in fetal life and is the only form of the enzyme till the 19th to 21st days of postnatal life when the second form of arginase, A4, appears and rapidly accumulates, being exclusively responsible for the rise of the total enzyme activity at the time of weaning. In kidney, arginase A1 alone is present in the early fetal period. Arginase A4 appears 3-4 days before birth and its activity persists unchanged within the first 2 weeks of postnatal life. The intensive rise in total specific activity of kidney arginase at weaning is due to the accumulation of preexisting arginase A4. In brain, the adult pattern of arginase isoenzymes is achieved earlier than in other tissues. Both forms, A1 and A4, occur on Days 13-14 of gestation.     

Arginine deprivation and tumour cell death: Arginase and its inhibition

Arginase treatment of cell cultures reduced arginine in the medium to ~ micromolar levels within 5–30 min, and proved as effective as arginine-free medium (AFM) prepared by formulation. The enzyme was heat stable and as active at pH 7.2 as at pH 9.9. It persisted in culture for at least 3 days with only a small diminution in its speed of action, and still actively destroyed arginine after 6 days, since arginine supplementation failed to rescue viable cells.Addition of L-norvaline, an inhibitor of arginase, rescued cells from arginase-induced deprivation. Its efficacy at low concentrations was short-lived (probably < 1 day), while at higher concentrations it did not appear to inhibit completely the enzyme. However, L-norvaline at these same levels also slowed the growth of positive non-enzyme treated controls receiving the normal arginine levels. Thus the difference in this growth indicated that arginase was more inhibitory than cursory examination of initial kinetic data suggested. It also agreed with the inhibition of arginase in the ornithine assay used to measure biochemically enzyme activity. We conclude that norvaline partially but not completely antagonises arginase activity, which allows cell rescue in a dose-dependent manner between 0.4 and 4 mM, but cannot be used above about 2 mM without exhibiting a general non-specific interference of cell growth of its own, although no evidence of cell toxicity was observed in either AFM or arginine-containing medium. L-ornithine, the product of arginase that inhibits the enzyme by a feedback mechanism, had no inhibitory effect on arginase over a similar concentration range.     

Time course of vascular arginase expression and activity in spontaneously hypertensive rats

There is growing evidence that vascular arginase plays a role in pathophysiology of vascular diseases. We recently reported high arginase activity/expression in young adult hypertensive spontaneously hypertensive rats (SHR). The aim of the present study was to characterize the time course of arginase pathway abnormalities in SHR and to explore the contributing role of hemodynamics and inflammation. Experiments were conducted on 5, 10, 19 and 26-week-old SHR and their age-matched control Wistar Kyoto (WKY) rats. Arginase activity as well as expression of arginase I, arginase II, endothelial and inducible NOS were determined in aortic tissue extracts. Levels of L-arginine, NO catabolites and IL-6 (a marker of inflammation) were measured in plasma. Arginase activity/expression was also measured in 10-week-old SHR previously treated with hydralazine (20 mg/kg/day, per os, for 5 weeks). As compared to WKY, SHR exhibited high vascular arginase I and II expression from prehypertensive to established stages of hypertension. However, a mismatch between expression and activity was observed at the prehypertensive stage. Arginase expression was not related either to plasma IL-6 levels or to expression of NOS. Prevention of hypertension by hydralazine significantly blunted arginase upregulation and restored arginase activity. Importantly, arginase activity and blood pressure (BP) correlated in SHR. In conclusion, our results demonstrate that arginase upregulation precedes blood pressure rising and identify elevated blood pressure as a contributing factor of arginase dysregulation in genetic hypertension. They also demonstrated a close relationship between arginase activity and BP, thus making arginase a promising target for antihypertensive therapy.     

In vivo treatment with progestogens causes immunosuppression of carp Cyprinus carpio leucocytes by affecting nitric oxide production and arginase activity

In this study, carp Cyprinus carpio were injected with various steroid compounds, including synthetic and natural progestogens and the glucocorticoid cortisol, to investigate effects on leucocytes isolated from their kidneys. Injection of cortisol led to an increased spleeno-somatic index (I(S)) on day 21 post-injection (pi) and immunosuppressive effects measured as decreased nitric oxide (NO) production and increased arginase activity in isolated leucocytes on days 14 and 21 pi, respectively. Moreover, reduced NO production was also observed after injection of the synthetic progestogens, levonorgestrel (LEV) and medroxyprogesterone acetate. In addition, LEV influenced arginase activity in head kidney cells on day 14 and day 21 pi. This study is the first demonstration in fishes that the application of these steroid compounds in vivo affects NO production and arginase activity of isolated leucocytes.     

Transiently, paralleled upregulation of arginase and nitric oxide synthase and the effect of both enzymes on the pathology of asthma

Changes in the expression of arginase and their association with nitrosative stress were investigated using an asthmatic model previously established in NC/Nga mice with mite extract. Mite crude extract (100 microg/day) from Dermatophagoides farinae was administered intranasally for 5 consecutive days (day 0-4), and a single challenge was performed on day 11. On day 12, upregulation of the mRNA expression of inducible types of nitric oxide synthase (iNOS) and increases in immunohistochemical staining for iNOS and nitrotyrosine were observed. However, the level of nitrite + nitrate was unchanged. An increase in enzymatic activity, upregulation of mRNA expression, and immunostaining for arginase I was detected in the lung tissue and serum. Moreover, increases in both arginase I and II were revealed by immunoblotting. Goblet cell hyperplasia in bronchial epithelial cells and increasing collagen synthesis around the bronchus were also observed. These results suggested that an increase in arginase may lead to decreased availability of arginine for nitric oxide synthase and may contribute to the remodeling of the lung.     

Tissue-specific differential modulation of arginase and ornithine aminotransferase by hydrocortisone during various developmental stages of the rat

The activities and regulatory patterns of arginase and ornithine aminotransferase (OAT) of the liver (a mitotic tissue) and kidney cortex (a post-mitotic tissue) of immature, adult, and senescent male rats were studied. The activities of the liver enzymes were highest in the immature rat and decreased gradually with age. However, in the kidney cortex, the activity of arginase was highest and decreased significantly thereafter while that of OAT shows no significant change throughout the life span of the rat. Further, the activity of kidney cortex arginase was approximately 1/20th of that of the liver enzyme. Adrenalectomy and hydrocortisone treatments altered the activity of arginase in both tissues and that of OAT in the liver only. However, the kidney cortex OAT was not responsive towards these treatments. Actinomycin D inhibited the hydrocortisone-mediated induction of arginase of both the liver and kidney cortex and that of the liver OAT.     

Purification and properties of Pinus taeda arginase from germinated seedlings

In germinated loblolly pine (Pinus taeda L.) seeds arginine accumulates in the seedling during its growth immediately following germination. The enzyme arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is responsible for hydrolyzing this arginine into ornithine and urea. Loblolly pine arginase was purified to homogeneity from seedling cotyledons by chromatographic separation on DE-52 cellulose, Matrex Green and arginine-linked Sepharose 4B. The enzyme was purified 148-fold and a single polypeptide band was identified as arginase. The molecular mass was determined to be 140 kDa by FPLC, while the subunit size was shown to be 37 kDa by SDS-PAGE, predicting a homotetramer holoprotein. Removal of manganese from the enzyme abolishes catalytic activity, which can be restored by incubating the protein with Mn²⁺. Antibodies, raised against the arginase subunit, are able to immunotitrate arginase activity and are monospecific for arginase on immunoblots.     

Angiotensin II-induced vascular endothelial dysfunction through RhoA/Rho kinase/p38 mitogen-activated protein kinase/arginase pathway

Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 μM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 μM). Blockade of ANG II AT(1) receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 μM) or Rho kinase (ROCK) (Y-27632, 10 μM; H1152, 0.5 μM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 μM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 μg·kg(-1)·day(-1)) or arginase (ABH, 8 mg·kg(-1)·day(-1)) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT(1) receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.     

Preparation of a non-immunogenic arginase by the covalent attachment of polyethylene glycol.

Methoxypolyethylene glycol of 5000 daltons (PEG) was attached covalently to bovine liver arginase using 2,4,6-trichloro-s-triazine as the coupling agent. The conjugate (PEG-arginase), with PEG attached to 53% of the amino groups, retained 65% of its original enzymatic activity. Mice were injected intravenously with arginase or PEG-arginase for periods of one to three months. The blood-circulating life of PEG-arginase was greatly extended over that of arginase. The half-life of injected arginase at day 30 was less than 1 h, whereas that of the PEG-enzyme was 12 h. Antisera from mice injected with native arginase reacted against arginase but not against PEG-arginase when tested by immunodiffusion. Antisera from animals injected with PEG-arginase reacted neither with native arginase nor PEG-arginase. The data indicate that arginase modified by PEG has been rendered both non-immunogenic and non-antigenic when tested in mice. The injection of PEG-arginase into mice did not induce tolerance toward the native enzyme. Injected PEG-arginase, in the presence of precipitating antibody directed against native arginase, circulated at the same level as in virgin animals. The attachment of PEG to arginase altered its kinetic properties.     

Inhibition of arginase ameliorates experimental ulcerative colitis in mice

Abstract

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

Comparison of biochemical properties of liver arginase from streptozocin-induced diabetic and control mice

Arginase activity is elevated in livers of diabetic animals compared to controls and there is evidence that this is due in part to increased specific activity (activity/mg arginase protein). To investigate the molecular basis of this increased activity, the physicochemical and kinetic properties of hepatic arginase from diabetic and control mice were compared. Two types of arginase subunits with molecular weights of 35,000 and 38,000 were found in both the diabetic and control animals and the subunits in these animals had similar, multiple ionic forms. Kinetic parameters of purified preparations of arginase for arginine (apparent Km and Vmax values) and the thermal stability of these preparations from diabetics and controls were also similar. Furthermore, no difference was found in the distribution of arginase activity among different subcellular liver fractions. Separation of basic and acidic oligomeric forms of arginase by fast-protein liquid chromatography resulted in a slightly different distribution of activity among the forms in the normal and diabetic group. The apparent Km values for Mn2+ of the basic form of the enzyme were 25 and 33 microM for the enzyme from normal and diabetic animals, respectively; for acidic forms, for which two apparent Km values were measured, the values were 8 and 197 microM for arginase from controls and 35 and 537 microM from diabetics. These results indicate that in diabetes, while no marked changes in the physicochemical characteristics of arginase are obvious, some changes are found in the interaction of arginase with its cofactor Mn.     

Inhibition of phosphodiesterase 4 amplifies cytokine-dependent induction of arginase in macrophages

Arginase is greatly elevated in asthma and is thought to play a role in the pathophysiology of this disease. As inhibitors of phosphodiesterase 4 (PDE4), the predominant PDE in macrophages, elevate cAMP levels and reduce inflammation, they have been proposed for use in treatment of asthma and chronic obstructive pulmonary disease. As cAMP is an inducer of arginase, we tested the hypothesis that a PDE4 inhibitor would enhance macrophage arginase induction by key cytokines implicated in asthma and other pulmonary diseases. RAW 264.7 cells were stimulated with IL-4 or transforming growth factor (TGF)-beta, with and without the PDE4 inhibitor rolipram. IL-4 and TGF-beta increased arginase activity 16- and 5-fold, respectively. Rolipram alone had no effect but when combined with IL-4 and TGF-beta synergistically enhanced arginase activity by an additional 15- and 5-fold, respectively. The increases in arginase I protein and mRNA levels mirrored increases in arginase activity. Induction of arginase II mRNA was also enhanced by rolipram but to a much lesser extent than arginase I. Unlike its effect in RAW 264.7 cells, IL-4 alone did not increase arginase activity in human alveolar macrophages (AM) from healthy volunteers. However, combining IL-4 with agents to induce cAMP levels induced arginase activity in human AM significantly above the level obtained with cAMP-inducing agents alone. In conclusion, agents that elevate cAMP significantly enhance induction of arginase by cytokines. Therefore, consequences of increased arginase expression should be evaluated whenever PDE inhibitors are proposed for treatment of inflammatory disorders in which IL-4 and/or TGF-beta predominate.     

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.     

Activities of glutamine synthetase,glutaminase and arginase in kainic acid lesioned rat brain corpus striatum

Intrastriatal kainic acid (2 μg/μl) administration gave rise to significant increase in activities of glutamine synthetase and arginase along with a significant decrease in the activity of glutaminase in the lesioned striatal tissue 7 days after the administration of kainic acid. The increase in the activity of glutamine synthetase was attributed to the gliosis occurring in such lesions. The decrease in the activity of glutaminase was thought to be due to the loss of GABAergic neurons. The increase in arginase activity might be occurring in glial cells or in nerve endings. Although the earlier results indicated a low specific activity of arginase in glial cells, the observed increase in its activity might be partly due to its increase in proliferating glial cells, liberating ornithine for the formation of polyamines. However, it was also thought that a substantial increase may be occurring in the arginase present in the intact glutamatergic (corticostriate pathway) nerve endings, since it was earlier found that the synaptosomes of the rat brain had appreciably high activity of arginase. These results were discussed in relation to the probable roles of arginine and glutamine as the precursors for neurotransmitter pools of glutamate in striatum.     

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.     

Studies on L-arginase in developing rat small intestine, brain, and kidney. II. Effect of hydrocortisone and thyroxine

The influences of hydrocortisone and thyroxine on the developmental changes of arginase activity in intestine, kidney, and brain of suckling rats were studied. A single injection of hydrocortisone (50 mg/kg) into rats aged 9 days evoked premature increase of jejunal arginase activity due to precocious formation of arginase A4. Arginase A4 can be detected about 48 hr after hydrocortisone injection, whereas in intact rats the enzyme appears in the intestinal mucosa on the 19th-21st days of postnatal life. After hydrocortisone administration to rats aged 6 days, a similar pattern of arginase activity in jejunum was observed. Under the same conditions, the influence of hydrocortisone on kidney arginase was weaker. The hormone did not have any influence on the activity of brain arginase. Daily injection of thyroxine (2 mg/kg) to 6-day-old rats (for 6 consecutive days) caused a precocious increase of the arginase activity in intestine. Under the same conditions, only a slight increase of the arginase activity was observed in kidney, whereas in brain the activity was unaffected.     

Inhibition of thyroxine-induced metamorphosis by prolactin in tadpoles, Rana catesbeiana.

Thyroxine (T4)-prolactin interactions on hepatic arginase and ornithine transcarbamylase (OTC) as well as hind legs, tail, digestive tract and median eminence were investigated in tadpoles, Rana catesbeiana. Prolactin completely blocked T4-induced tail resorption, but failed to suppress hind-leg growth, shortening of digestive tract and promotion by T4 of the median eminence development. Prolactin blocked T4-induced increase in hepatic arginase activity but not in hepatic OTC activity. A possibility that T4 and prolactin are regulating the hepatic arginase indirectly is discussed.     

Biochemical and molecular characterization of mitochondrial membrane-bound arginase in <Emphasis Type="Italic">Heteropneustes fossilis</Emphasis>

The two predominant forms of arginase, cytosolic Arginase-I and mitochondrial Arginase-II, catalyze hydrolysis of arginine into ornithine and urea. Based on presence of arginase activity in extracts using potassium chloride (KCl), mitochondrial membrane-bound arginase has also been suggested. However, the activity of arginase in fractions obtained after KCl-treatment may be either due to leakage of mitochondrial arginase or release of adhered cytosolic arginase to cell organelles having altered net charge. Therefore, it has been intended to analyse impact of KCl on ultra-structural properties of mitochondria, and biochemical analysis of mitochondrial membrane-bound proteins and arginase of Heteropneustes fossilis. Liver of H. fossilis was used for isolating mitochondria for analysis of ultrastructural properties, preparing cytosolic, mitochondrial, and mitochondrial-membrane bound extracts after treatment of KCl. Extracts were analysed for arginase activity assay, protein profiling through SDS-PAGE and MALDI MS/MS. The KCl-mediated modulation in polypeptides and arginase were also evaluated by PANTHER, MitoProt and IPSORT servers. The effects of KCl on ultra-structural integrity of mitochondria, activity of arginase, modulation on mitochondrial proteins and enzymes including arginase were observed. The 48 kDa polypeptide of mitochondrial fraction, that showed KCl-dependent alteration matched with Myb binding protein and 30 kDa bands resembles to arginase after MALDI MS/MS analysis. Results indicate KCl-dependent ultrastructural changes in mitochondria and release of mitochondrial arginase. The proposed membrane bound mitochondrial arginase could be mitochondrial arginase-II or altered form of cytosolic arginase-I contributing to KCl-induced arginase activity in H. fossilis.     

Developmental changes in arginase expression and activity in the lung

Arginases compete with nitric oxide (NO) synthases for L-arginine as common substrate. Pulmonary vascular and airway diseases in which arginase activity is increased are associated with decreased NO production and reduced smooth muscle relaxation. The developmental patterns of arginase activity and type I and II isoforms expression in the lung have not been previously evaluated. Hypothesizing that lung arginase activity is developmentally regulated and highest in the fetus, we measured the expression of both arginase isoforms and total arginase activity in fetal, newborn, and adult rat lung, pulmonary artery, and bronchial tissue. In addition, intrapulmonary arterial muscle force generation was evaluated in the absence and presence of the arginase inhibitor Nomega-hydroxy-nor-L-arginine (nor-NOHA). Arginase II content, as well as total arginase activity, was highest in fetal rat lung, bronchi, and pulmonary arterial tissue and decreased with age (P<0.05), and its lung cell expression was developmentally regulated. In the presence of nor-NOHA, pulmonary arterial force generation was significantly reduced in fetus and newborn (P<0.01). No significant change in force generation was noted in bronchial tissue following arginase inhibition. In conclusion, arginase II is regulated developmentally, and both expression and activity are maximal during fetal life. We speculate that the maintenance of a high pulmonary vascular resistance and decreased lung NO production prenatally may, in part, be dependent on increased arginase expression and/or activity.