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.     

Heterogeneity of glycogen synthesis upon refeeding following starvation.

1. Starvation of rats for 40 hr decreased the body weight, liver weight and blood glucose concentration. The hepatic and skeletal muscle glycogen concentrations were decreased by 95% (from 410 mumol/g tissue to 16 mumol/g tissue) and 55% (from 40 mumol/g tissue to 18.5 mumol/g tissue), respectively. 2. Fine structural analysis of glycogen purified from the liver and skeletal muscle of starved rats suggested that the glycogenolysis included a lysosomal component, in addition to the conventional phosphorolytic pathway. In support of this the hepatic acid alpha-glucosidase activity increased 1.8-fold following starvation. 3. Refeeding resulted in liver glycogen synthesis at a linear rate of 40 mumol/g tissue per hr over the first 13 hr of refeeding. The hepatic glycogen store were replenished by 8 hr of refeeding, but synthesis continued and the hepatic glycogen content peaked at 24 hr (approximately 670 mumol/g tissue). 4. Refeeding resulted in skeletal muscle glycogen synthesis at an initial rate of 40 mumol/g tissue per hr. The muscle glycogen store was replenished by 30 min of refeeding, but synthesis continued and the glycogen content peaked at 13 hr (approximately 50 mumol/g tissue). 5. Both liver and skeletal muscle glycogen synthesis were inhomogeneous with respect to molecular size; high molecular weight glycogen was initially synthesised at a faster rate than low molecular weight glycogen. These observations support suggestions that there is more than a single site of glycogen synthesis.     

Determination of isocitrate lyase and malate synthase activities in a marine bivalve mollusk by a new method of assay

1. A new method for the assay of isocitrate lyase (EC 4.1.3.1) was developed, based on the isolation of 14C-glyoxylate semicarbazone by co-crystallization with authentic carrier. The method was easily adapted to measure malate synthase (EC 4.1.3.2). 2. Interfering reactions were avoided with this method, and isocitrate dehydrogenase (ID) was easily distinguished from isocitrate lyase (IL). Assay of IL in germinating pumpkin seeds gave rates proportional to the amount of extract, with greater sensitivity and less variability than the spectrophotometric method. 3. Six species of marine bivalve mollusks were tested for IL activity, and two species produced glyoxylate: Crassostrea virginica at 0.10 mumol/hr/g tissue, and Petricola pholadiformis at 0.85 mumol/hr/g. The other four species, and four other marine invertebrates from other phyla, lacked detectable IL activity. 4. The rate of disappearance of glyoxylate in the malate synthase (MS) reaction indicated that Petricola had an activity of 0.60 mumol/hr/g: this is the first demonstration of activities of both IL and MS in a marine invertebrate.     

Dopamine-beta-hydroxylase activity in the axillary bodies, the heart and the splanchnic nerve in two elasmobranchs, Squalus acanthias and Etmopterus spinax

1. The activity of dopamine-beta-hydroxylase (DBH) was examined in tissues from Squalus acanthias and Etmopterus spinax using tyramine as substrate. 2. The activity of DBH in axillary bodies in Squalus was 19040 +/- 240 nmol/g per hr and in Etmopterus 6120 +/- 245 nmol/g per hr. 3. DBH activity in nervous tissue, i.e. the splanchnic nerve (together with coeliac artery) was found to be 123 +/- 41 nmol/g per hr in Squalus and 384 +/- 62 nmol/g per hr in Etmopterus. 4. In Squalus heart DBH activity was undetectable, while Etmopterus heart had DBH activity both in atrium and ventricle, 40 +/- 13 and 18 +/- 8 nmol/g per hr respectively. 5. A small amount of DBH activity was found in Squalus blood plasma, 2 +/- 0.7 nmol/ml per hr. 6. The DBH activity gave high formation of octopamine in a wide temperature range from 24.5 to 32 degrees C.     

Inhibition of arginase activity enhances inflammation in mice with allergic airway disease, in association with increases in protein S-nitrosylation and tyrosine nitration

Pulmonary inflammation in asthma is orchestrated by the activity of NF-kappaB. NO and NO synthase (NOS) activity are important modulators of inflammation. The availability of the NOS substrate, l-arginine, is one of the mechanisms that controls the activity of NOS. Arginase also uses l-arginine as its substrate, and arginase-1 expression is highly induced in a murine model of asthma. Because we have previously described that arginase affects NOx content and interferes with the activation of NF-kappaB in lung epithelial cells, the goal of this study was to investigate the impact of arginase inhibition on the bioavailability of NO and the implications for NF-kappaB activation and inflammation in a mouse model of allergic airway disease. Administration of the arginase inhibitor BEC (S-(2-boronoethyl)-l-cysteine) decreased arginase activity and caused alterations in NO homeostasis, which were reflected by increases in S-nitrosylated and nitrated proteins in the lungs from inflamed mice. In contrast to our expectations, BEC enhanced perivascular and peribronchiolar lung inflammation, mucus metaplasia, NF-kappaB DNA binding, and mRNA expression of the NF-kappaB-driven chemokine genes CCL20 and KC, and lead to further increases in airways hyperresponsiveness. These results suggest that inhibition of arginase activity enhanced a variety of parameters relevant to allergic airways disease, possibly by altering NO homeostasis.     

31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg2+ in human erythrocytes

Absolute 31P-NMR measurements of ATP, ADP and 2,3-diphosphoglycerate (2,3-DPG) in oxygenated and partly deoxygenated human erythrocytes, compared to measurements by standard assays after acid extraction, show that ATP is only 65% NMR visible, ADP measured by NMR is unexpectedly 400% higher than the enzymatic measurement and 2,3-DPG is fully NMR visible, regardless of the degree of oxygenation. These results show that binding to hemoglobin is unlikely to cause the decreased visibility of ATP in human erythrocytes as deoxyhemoglobin binds the phosphorylated metabolites more tightly than oxyhemoglobin. The high ADP visibility is unexplained. The levels of free Mg2+ [( Mg2+]free) in human erythrocytes are 225 mumol/l at an oxygen saturation of 98.6% and instead of the expected increase, the level decreased to 196 mumol/l at an oxygen saturation of 38.1% based on the separation between the alpha- and beta-ATP peaks. [Mg2+]free in the erythrocytes decreased to 104 mumol/l at a high 2,3-DPG concentration of 25.4 mmol/l red blood cells (RBC) and a normal ATP concentration of 2.05 mmol/l RBC. By increasing the ATP concentration to 3.57 mmol/l RBC, and with a high 2,3-DPG concentration of 24.7 mmol/l RBC, the 31P-NMR measured [Mg2+]free decreased to 61 mumol/l. These results indicate, that the 31P-NMR determined [Mg2+]free in human erythrocytes, based solely on the separation of the alpha- and beta-ATP peaks, does not give a true measure of intracellular free Mg2+ changes with different oxygen saturation levels. Furthermore the measurement is influenced by the concentration of the Mg2+ binding metabolites ATP and 2,3-DPG. Failure to take these factors into account when interpreting 31P-NMR data from human erythrocytes may explain some discrepancies in the literature regarding [Mg2+]free.     

Erythrocyte phosphate composition and osmotic fragility in the Australian lungfish, Neoceratodus fosteri, and osteoglossid, Scleropages schneichardti

The packed cell volume (PCV), hemoglobin concentration (g/dl) and mean corpuscular volume (MCV) in the Australian lungfish, Neoceratodus fosteri, and in one of three Australian osteoglossids, Scleropages schneichardti, were 32.3 and 29.9; 10.5 and 10.0; and 407 and 176 micron 3 respectively. Total acid-soluble phosphates (TPi) from the red blood cells (RBC) of the lungfish and osteoglossid were 35.3 and 18.1 mumol/cm3 RBC respectively. Inorganic phosphate (Pi), adenosine triphosphate (ATP) and guanosine triphosphate (GTP) represented 16.4, 39.7 and 17.8% of the cell phosphates in the lungfish respectively. Inositol bisphosphate was not present in extracts of the red cells of N. fosteri, in contrast to the red cells of Lepidosiren paradoxa and Protopterus aethiopicus, in which it was first observed. In the osteoglossid, Pi and ATP represented 37.6 and 46.4% of the erythrocyte phosphate, respectively, with only traces of GTP present. ATP is the predominant organic phosphate in the red cells of both species. The osmotic fragility of erythrocytes of N. fosteri are quite resistant to hemolysis, with hemolysis beginning at 35-30 mM and a complete hemolysis occurring at 20 mM NaCl. The red cells of S. schneichardti begin to hemolyze at 95-90 mM with hemolysis continuing to completion at 60 mM NaCl.     

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.     

Roles of accumulated endogenous nitric oxide synthase inhibitors, enhanced arginase activity, and attenuated nitric oxide synthase activity in endothelial cells for pulmonary hypertension in rats

Nitric oxide (NO) has been suggested to play a key role in the pathogenesis of pulmonary hypertension (PH). To determine which mechanism exists to affect NO production, we examined the concentration of endogenous nitric oxide synthase (NOS) inhibitors and their catabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) activity and protein expression (DDAH1 and DDAH2) in pulmonary artery endothelial cells (PAECs) of rats given monocrotaline (MCT). We also measured NOS and arginase activities and NOS protein expression. Twenty-four days after MCT administration, PH and right ventricle (RV) hypertrophy were established. Endothelium-dependent, but not endothelium-independent, relaxation and cGMP production were significantly impaired in pulmonary artery specimens of MCT group. The constitutive NOS activity and protein expression in PAECs were significantly reduced in MCT group, whereas the arginase, which shares l-arginine as a common substrate with NOS, activity was significantly enhanced in PAECs of MCT group. The contents of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA), were increased in PAECs of MCT group. The DDAH activity and DDAH1, but not DDAH2, protein expression were significantly reduced in PAECs of MCT group. These results suggest that the impairment of cGMP production as a marker of NO production is possibly due to the blunted endothelial NOS activity resulting from the downregulation of endothelial NOS protein, accumulation of endogenous NOS inhibitors, and accelerated arginase activity in PAECs of PH rats. The decreased overall DDAH activity accompanied by the downregulation of DDAH1 would bring about the accumulation of endogenous NOS inhibitors.     

In vivo dose response relationship between physostigmine and cholinesterase activity in RBC and tissues of rats

Dose response of physostigmine (Phy) was studied in rat using various doses (25-500 micrograms/kg i.m.). Rats were sacrificed 15 min after Phy administration. Blood and tissues were analyzed for ChE activity by radiometric method and Phy concentration by HPLC method. A comparison of ChE values in different tissues of rats indicated that ChE activity was highest in brain (7.11 mumol/min/g) and least in diaphragm (0.67 mumol/min/g). The enzyme activity was eleven times more in brain as compared to diaphragm. Phy produced a dose-dependent inhibition of ChE in RBC (18-42%), brain (23-35%) and diaphragm (25-35%) from 50 to 200 micrograms/kg, then ChE inhibition was plateaued from 200 to 500 micrograms/kg in these tissues. A dose related ChE inhibition was seen in heart (16-50%) and thigh muscle (8-53%) from 50 to 500 micrograms/kg. Phy concentration increased linearly from 50 to 400 micrograms/kg in plasma, brain, heart and thigh muscle. These results indicate that ChE inhibition is linear up to 200 micrograms/kg in RBC, 150 micrograms/kg in brain and 300 micrograms/kg in heart. This linearity is not consistent in other tissues.     

Cocoa flavanols lower vascular arginase activity in human endothelial cells in vitro and in erythrocytes in vivo

The availability of l-arginine can be a rate-limiting factor for cellular NO production by nitric oxide synthases (NOS). Arginase competes with NOS for l-arginine as the common substrate. Increased arginase activity has been linked to low NO levels, and an inhibition of arginase activity has been reported to improve endothelium-dependent vasorelaxation. Based on the above, we hypothesized that an increase in the circulating NO pool following flavanol consumption could be correlated with decreased arginase activity. To test this hypothesis we (a) investigated the effects of (−)-epicatechin and its structurally related metabolites on endothelial arginase expression and activity in vitro; (b) evaluated the effects of dietary flavanol-rich cocoa on kidney arginase activity in vivo; and (c) assessed human erythrocyte arginase activity following flavanol-rich cocoa beverage consumption in a double-blind intervention study with cross-over design. The results demonstrate that cocoa flavanols lower arginase-2 mRNA expression and activity in HUVEC. Dietary intervention with flavanol-rich cocoa caused diminished arginase activity in rat kidney and, erythrocyte arginase activity was lowered in healthy humans following consumption of a high flavanol beverage in vivo.     

Properties of arginase from liver of Macaca fascicularis: Comparison of normals with red blood cell arginase deficient monkeys

Deficiency of arginase (E.C. 3.5.3.1), the fifth enzyme of the urea cycle, was found in the red blood cells (RBCs) of Macaca fascicularis monkeys (<0.2 µmol arginine cleaved/g Hb/min; normal =49.2). Liver biopsies were obtained from two of these monkeys and from one monkey with normal levels of RBC arginase activity. Arginase from both groups of animals required Mn²⁺ for maximal enzyme activity and demonstrated a pH optimum of 10.2 in vitro. The activity of arginase in the livers of all three monkeys was 1.1 mmol arginine cleaved/g protein/min. The apparent K _m for arginine of arginase in the livers of the RBC-deficient monkeys was 7.4 and 5.9mm and in the normal monkey was 6.9mm. Similar patterns of heat denaturation were seen at 69 C without Mn²⁺ present and 79 C in the presence of 20mm Mn²⁺. No difference in mobility on either acidic or basic polyacrylamide gels for liver arginase from either RBC-deficient or normal monkeys was found. In addition, liver arginase from all three monkeys reacted similarly with anti-human liver arginase antibody. Liver arginases in RBC-deficient and normal monkeys were identical by ten criteria. These studies do not distinguish among several hypotheses for the genetic determination of arginase in different organs of this species and of man.     

3,4-Dihydroxyphenylalanine (dopa) decarboxylase activity in the arthropod nervous system

1. When homogenates of brains from mature adult locusts (Locusta migratoria) were incubated with l-3-(3,4-dihydroxyphenyl)[3-(14)C]alanine the major radioactive metabolite was dopamine, suggesting the presence of a dopa (3,4-dihydroxyphenylalanine) decarboxylase. 2. Decarboxylation of l-dopa by this tissue, measured under optimum conditions by a radiochemical method, was 21mumol of CO(2)/h per g wet wt. Apparent decarboxylation of l-tyrosine proceeded at 0.34mumol of CO(2)/h per g wet wt. There was no detectable decarboxylation of l-tryptophan, l-histidine or l-phenylalanine. 3. Dopa decarboxylase activity was found in all major regions of the ventral nerve cord of the mature locust (range: 4-7mumol of CO(2)/h per g wet wt.) but was low or absent in thoracic peripheral nerve. 4. Marked decarboxylation of l-dopa was found in homogenates of brains of four other species of insects, and in brain and ventral nerve cord, but not in the claw nerve, of the crayfish. 5. The activity of the locust brain enzyme may be slightly lower at the time of imaginal ecdysis than during the mature period. By contrast, the dopa decarboxylase that produces dopamine as an intermediate in cuticle biosynthesis is known to be high in activity at the time of ecdysis and low in activity during the intermoult stages.     

Arginase,an s-phase enzyme in a human cell line

Suspension cultures of ‘Chang liver’ cells were synchronized by preincubation in a glutamine-deficient medium or by thymidine blockade. Specific arginase activity varied in the synchronized cultures, being high when the number of S-phase cells was maximal. A relationship between high arginase activity and a high percentage of (S+G₂) cells was also found when unsynchronized cells were separated by velocity sedimentation. The increase in arginase activity near the G₁/S border was totally inhibited in the presence of cycloheximide. The rate of decrease in activity after addition of the drug indicated that the variations in the rate of synthesis of the enzyme, while the rate of degradation was more or less constant, corresponding to 4–6% per h. The role of arginase in cells lacking a urea cycle and the regulation of arginase activity in ‘Chang liver’ cells is discussed.     

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.     

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.     

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.     

Involvement of altered arginase activity, arginase I expression and NO production in accelerated intimal hyperplasia following cigarette smoke extract

In the present experiments, we tried to elucidate whether changes in arginase activity, protein expression of arginase-I and -II, and NO production are involved in accelerating the intimal hyperplasia following administration of cigarette smoke extract (CSE). The intimal hyperplasia was caused by removing endothelial cells with the aid of balloon embolectomy catheter in the right carotid artery of the male rabbit. The left carotid artery underwent sham operation and served as control. CSE was prepared by bubbling a stream of cigarette smoke into phosphate buffered saline. Rabbits were given subcutaneously with CSE once a day for 5 weeks from 1 week before to 4 weeks after the surgery. The specimens were assessed histologically and the intima/media ratio (%) was evaluated as an index of the intimal hyperplasia. The accelerated intimal hyperplasia with CSE was accompanied by the augmentation of the impaired cyclic GMP production, enhanced overall arginase activity and up-regulation of arginase-I. Pearson's correlation coefficient analyses revealed the close relationships among the arginase activities in endothelial cells and smooth muscle layer, the intimal/media ratio and cyclic GMP production. These results suggest that the enhanced arginase activity together with facilitated up-regulation of arginase-I with CSE, which was associated with the augmented impairment of NO production, shed a new light on the processes associated with accelerating the intimal hyperplasia in rabbit carotid arteries following CSE.     

Mitochondrial arginase II constrains endothelial NOS-3 activity

Emerging evidence supports the idea that arginase, expressed in the vascular endothelial cells of humans and other species, modulates endothelial nitric oxide (NO) synthase-3 (NOS-3) activity by regulating intracellular L-arginine bioavailability. Arginase II is thought to be expressed in the mitochondria of a variety of nonendothelial cells, whereas arginase I is known to be confined to the cytosol of hepatic and other cells. The isoforms that regulate NOS-3 and their subcellular distribution, however, remain incompletely characterized. We therefore tested the hypothesis that arginase II is confined to the mitochondria and that mitochondrial arginase II reciprocally regulates vascular endothelial NO production. Western blot analysis, immunocytochemistry with MitoTracker, and immunoelectron microscopy confirmed that arginase II is confined predominantly but not exclusively to the mitochondria. Arginase activity was significantly decreased, whereas NO production was significantly increased in the aorta and isolated endothelial cells from arginase II knockout (ArgII(-/-)) mice compared with wild-type (WT) mice. The vasorelaxation response to acetylcholine (ACh) was markedly enhanced and the vasoconstrictor response to phenylephrine (PE) attenuated in ArgII(-/-) in pressurized mouse carotid arteries. Furthermore, inhibition of NOS-3 by N(G)-nitro-L-arginine methyl ester (L-NAME) impaired ACh response and restored the PE response to that observed in WT vessels. Vascular stiffness, as assessed by pulse wave velocity (PWV), was significantly decreased in ArgII(-/-) compared with WT mice. On the other hand, 14 days of oral L-NAME treatment significantly increased PWV in both WT and ArgII(-/-) mice, such that they were not significantly different from one another. These data suggest that arginase II is predominantly confined to the mitochondria and that this mitochondrial arginase II regulates NO production, vascular endothelial function, and vascular stiffness by modulating NOS-3 activity.     

A colorimetric microplate assay method for high-throughput analysis of arginase activity in vitro

Several analytical methods have been developed for the determination of arginase (l-arginine amidinohydrolase) activity in physiological samples. These methods are limited by the considerable effort and time required to obtain reliable and reproducible measurements. Here we describe a simple high-throughput colorimetric assay for the determination of arginase activity based on the ornithine-ninhydrin reaction. This method is an improvement over the original single cuvette assay developed by Chinard in that no boiling step is required. The turnaround time has been reduced, with improved precision and reproducibility. The method was extended to the determination of arginase activity in human leukemic (K562) cells and sickle erythrocytes. We believe that the method will find applications for routine analysis as well as for characterizing the action of novel and potent inhibitors on arginase activity.