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Endothelial dysfunction, characterized by reduced bioavailability of nitric oxide and increased oxidative stress, is a hallmark characteristic in diabetes and diabetic nephropathy (DN). High levels of asymmetric dimethylarginine (ADMA) are observed in several diseases including DN and are a strong prognostic marker for cardiovascular events in patients with diabetes and end-stage renal disease. ADMA, an endogenous endothelial nitric oxide synthase (NOS3) inhibitor, is selectively metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Low DDAH levels have been associated with cardiac and renal dysfunction, but its effects on DN are unknown. We hypothesized that enhanced renal DDAH-1 expression would improve DN by reducing ADMA and restoring NOS3 levels. DBA/2J mice injected with multiple low doses of vehicle or streptozotocin were subsequently injected intrarenally with adenovirus expressing DDAH-1 (Ad-h-DDAH-1) or vector control [Ad-green fluorescent protein (GFP)], and mice were followed for 6 wk. Diabetes was associated with increased kidney ADMA and reduced kidney DDAH activity and DDAH-1 expression but had no effect on kidney DDAH-2 expression. Ad-GFP-treated diabetic mice showed significant increases in albuminuria, histological changes, glomerular macrophage recruitment, inflammatory cytokine and fibrotic markers, kidney ADMA levels, and urinary thiobarbituric acid reactive substances excretion as an indicator of oxidative stress, along with a significant reduction in kidney DDAH activity and kidney NOS3 mRNA compared with normal mice. In contrast, Ad-h-DDAH-1 treatment of diabetic mice reversed these effects. These data indicate, for the first time, that DDAH-1 mediates renal tissue protection in DN via the ADMA-NOS3-interaction. Enhanced renal DDAH-1 activity could be a novel therapeutic tool for treating patients with diabetes.

Mammalian arginine metabolism is complex due to the expression of multiple enzymes that utilize arginine as substrate and to interactions or competition between specific enzymes involved in arginine metabolism. Moreover, cells may contain multiple intracellular arginine pools that are not equally accessible to all arginine metabolic enzymes, thus presenting additional challenges to more fully understanding arginine metabolism. At the whole-body level, arginine metabolism ultimately results in the production of a biochemically diverse range of products, including nitric oxide, urea, creatine, polyamines, proline, glutamate, agmatine, and homoarginine. Included in this group of compounds are the methylated arginines (e.g., asymmetric dimethylarginine), which are released upon degradation of proteins containing methylated arginine residues. Changes in arginine concentration also can regulate cellular metabolism and function via a variety of arginine sensors. Although much is known about arginine metabolism, elucidation of the physiologic or pathophysiologic roles for all of the pathways and their metabolites remains an active area of investigation, as exemplified by current findings highlighted in this review., (© 2016 American Society for Nutrition.)

On the basis of research presented during the 9th Amino Acid Assessment Workshop, a No Observed Adverse Effect Level (NOAEL) for diet-added arginine (added mostly in the form of dietary supplements) of 30 g/d and an upper limit of safe intake (ULSI) for diet-added tryptophan (added mostly in the form of dietary supplements) of 4.5 g/d have been proposed. Both recommendations apply to healthy young adults. The total dietary leucine ULSI proposed for elderly individuals is 500 mg · kg -1 · d -1 All 3 recommendations are relevant only to high-quality amino acid-containing products with specifications corresponding to those listed in the US Pharmacopeia Because the above amino acids are extensively utilized as dietary supplements for various real or perceived benefits, such as vasodilation, spermatogenesis, sleep, mood regulation, or muscle recovery, the above safety recommendations will have an important impact on regulatory and nutritional practices., (© 2016 American Society for Nutrition.)

Our recent publication showed that pharmacological blockade of arginases confers kidney protection in diabetic nephropathy via a nitric oxide (NO) synthase (NOS)3-dependent mechanism. Arginase competes with endothelial NOS (eNOS) for the common substrate L-arginine. Lack of L-arginine results in reduced NO production and eNOS uncoupling, which lead to endothelial dysfunction. Therefore, we hypothesized that L-arginine or L-citrulline supplementation would ameliorate diabetic nephropathy. DBA mice injected with multiple low doses of vehicle or streptozotocin (50 mg/kg ip for 5 days) were provided drinking water with or without L-arginine (1.5%, 6.05 g·kg(-1)·day(-1)) or L-citrulline (1.66%, 5.73 g·kg(-1)·day(-1)) for 9 wk. Nonsupplemented diabetic mice showed significant increases in albuminuria, blood urea nitrogen, glomerular histopathological changes, kidney macrophage recruitment, kidney TNF-α and fibronectin mRNA expression, kidney arginase activity, kidney arginase-2 protein expression, and urinary oxidative stress along with a significant reduction of nephrin and eNOS protein expression and kidney nitrite + nitrate compared with normal mice after 9 wk of diabetes. Surprisingly, L-arginine or L-citrulline supplementation in diabetic mice did not affect any of these parameters despite greatly increasing kidney and plasma arginine levels. These findings demonstrate that chronic L-arginine or L-citrulline supplementation does not prevent or reduce renal injury in a model of type 1 diabetes., (Copyright © 2014 the American Physiological Society.)

Objective: To explore the hypothesis that decreased arginine availability by myeloid-derived suppressor cells (MDSCs) is a cause of T-cell dysfunction after physical injury (PI)., Background: Arginine is an essential amino acid for normal T-cell function whose availability becomes limited after PI. MDSCs expressing arginase 1 are induced by PI. T-cell dysfunction after PI seems to increase the risk of infection but the mechanisms that cause it are unclear., Methods: PI was created using a standard laparotomy model. Phenotypical and functional alterations in T cells were evaluated in vivo. MDSCs expressing arginase 1 were measured by flow cytometry. Infection after PI was created by intraperitoneal injection of Listeria monocytogenes. N-Hydroxy-Nor-L-arginine (Nor-NOHA) was used as an arginase inhibitor. The effect of arginine depletion on T-cell function and susceptibility to infection was assessed through adoptive transfer of MDSC or injection of arginase into noninjured mice., Results: PI caused a decrease in intracellular arginine in T cells, loss of the T-cell receptor (TCR) CD3-ζ chain, inhibition of in vivo T-cell proliferation, memory, and cytotoxicity. PI exponentially increased bacterial growth and mortality to L. monocytogenes. T-cell dysfunction and increased infection were reversed by arginase inhibitor Nor-NOHA but were reproduced by adoptively transferring MDSC or injecting arginase 1 to noninjured mice., Conclusions: Arginine availability is decreased after PI coinciding with an induction of MDSC expressing arginase 1. Decreased arginine may inhibit T-cell function and increase susceptibility to infection after injury.

Purpose of Review: Many physiologic and pathophysiologic processes are modulated by arginine availability, which can be regulated by arginase. An understanding of the conditions that result in elevated arginase activity as well as the consequences of arginine deficiency is essential for design of effective nutritional support for disease. This review will emphasize recent findings regarding effects of plasma arginase and arginine deficiencies in disease., Recent Findings: Elevations in plasma arginase, derived primarily from hemolysis of red blood cells or liver damage, that are associated with arginine deficiency have been identified in an increasing number of diseases and conditions. Arginine insufficiency not only can activate a stress kinase pathway that impairs function of T lymphocytes but it also can inhibit the mitogen-activated protein kinase signaling pathway required for macrophage production of cytokines in response to bacterial endotoxin/lipopolysaccharide., Summary: There are at least two broad categories of arginine deficiency syndromes, involving either T-cell dysfunction or endothelial dysfunction, depending on the disease context in which arginine deficiency occurs. There is limited information regarding the safety and efficacy of supplementation with arginine or its precursor citrulline in ameliorating arginine deficiency in specific diseases, indicating the need for further studies.

In healthy, mostly normotensive blacks, 19 salt-sensitive (SS) and 18 salt-resistant (SR), we tested the hypothesis that, in SS subjects, dietary NaCl loading induces its initial pressor effect by inducing a normal increase of cardiac output, while failing to induce a normal pressor-offsetting vasodilatation, consequent to its inhibition by asymmetrical dimethylarginine that is abnormally increased by NaCl. In SS and SR subjects, dietary NaCl loading, 250 from 30 mmol/d, over a 7-day period, induced similar, immediate increases in external Na(+) balance (by day 2, ≈360 mmol), plasma volume (+11%), and cardiac output (+8%). In SR subjects, from day 1, transient decreases occurred in both systemic vascular resistance (nadir: -13%, day 2) and mean arterial pressure (nadir: -5%, day 2). In SS subjects, systemic vascular resistance did not change over days 1 to 3, whereas mean arterial pressure increased progressively after day 1, ultimately by 10 mm Hg. Failure of systemic vascular resistance to normally decrease, while cardiac output normally increased, accounted for salt's initial pressor effect in the SS subjects. In SS subjects, baseline plasma levels of asymmetrical dimethylarginine (0.76 μmol/L) and symmetrical dimethylarginine (0.60 μmol/L), which does not affect vasodilatation, approximated those in SR subjects. In SS but not SR subjects, NaCl loading induced increases in asymmetrical dimethylarginine on both days 2 (+38%, median) and 7 (+14%, median). Symmetrical dimethylarginine changed in neither group. For all of the subjects combined, changes in asymmetrical dimethylarginine on day 2 predicted changes in systemic vascular resistance (R=0.751; P<0.001) and mean arterial pressure (R=0.527; P=0.006) on day 2 and similarly on day 7. These observations support the hypothesis tested.

Vascular disease, a significant cause of morbidity and mortality in the developed world, results from vascular injury. Following vascular injury, damaged or dysfunctional endothelial cells and activated SMCs engage in vasoproliferative remodeling and the formation of flow-limiting intimal hyperplasia (IH). We hypothesized that vascular injury results in decreased bioavailability of NO secondary to dysregulated arginine-dependent NO generation. Furthermore, we postulated that nitrite-dependent NO generation is augmented as an adaptive response to limit vascular injury/proliferation and can be harnessed for its protective effects. Here we report that sodium nitrite (intraperitoneal, inhaled, or oral) limited the development of IH in a rat model of vascular injury. Additionally, nitrite led to the generation of NO in vessels and SMCs, as well as limited SMC proliferation via p21Waf1/Cip1 signaling. These data demonstrate that IH is associated with increased arginase-1 levels, which leads to decreased NO production and bioavailability. Vascular injury also was associated with increased levels of xanthine oxidoreductase (XOR), a known nitrite reductase. Chronic inhibition of XOR and a diet deficient in nitrate/nitrite each exacerbated vascular injury. Moreover, established IH was reversed by dietary supplementation of nitrite. The vasoprotective effects of nitrite were counteracted by inhibition of XOR. These data illustrate the importance of nitrite-generated NO as an endogenous adaptive response and as a pathway that can be harnessed for therapeutic benefit.

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.

The activation of macrophages through Toll-like receptor (TLR) signaling pathways is a major component of innate immune responses to infection. Because the production of nitric oxide (NO) from arginine by the inducible isoform of NO synthase (iNOS) in activated macrophages is essential for host defense against many pathogens, arginine availability is a critical determinant of resistance to infection. Thus, induction of the arginine catabolic enzyme arginase is exploited by some pathogens as a means of immune evasion. Details of this mechanism are revealed by studies that demonstrate that mycobacteria use a component of the TLR pathway to induce the type I isoform of arginase in macrophages through an autocrine-paracrine mechanism that involves macrophage-produced cytokines. Separate studies show that, in addition to inhibiting NO synthesis by substrate limitation, reducing the availability of arginine simply by nutrient deprivation can blunt the innate immune response by impairing a specific mitogen-activated protein kinase (MAPK) pathway downstream of TLR4. These findings illustrate the growing complexity of the roles of arginine as an enzyme substrate and also as a regulatory molecule in signal transduction pathways in immune cells.

As arginine can serve as precursor to a wide range of compounds, including nitric oxide, creatine, urea, polyamines, proline, glutamate and agmatine, there is considerable interest in elucidating mechanisms underlying regulation of its metabolism. It is now becoming apparent that the two isoforms of arginase in mammals play key roles in regulation of most aspects of arginine metabolism in health and disease. In particular, work over the past several years has focused on the roles and regulation of the arginases in vascular disease, pulmonary disease, infectious disease, immune cell function and cancer. As most of these topics have been considered in recent review articles, this review will focus more closely on results of recent studies on expression of the arginases in endothelial and vascular smooth muscle cells, post-translational modulation of arginase activity and applications of arginase inhibitors in vivo.

Arginine has multiple metabolic fates and thus is one of the most versatile amino acids. Not only is it metabolically interconvertible with the amino acids proline and glutamate, but it also serves as a precursor for synthesis of protein, nitric oxide, creatine, polyamines, agmatine, and urea. These processes do not all occur within each cell but are differentially expressed according to cell type, age and developmental stage, diet, and state of health or disease. Arginine metabolism also is modulated by activities of various transporters that move arginine and its metabolites across the plasma and mitochondrial membranes. Moreover, several key enzymes in arginine metabolism are expressed as multiple isozymes whose expression can change rapidly and dramatically in response to a variety of different stimuli in health and disease. As illustrated by the questions raised in this article, we currently have an imperfect and incomplete picture of arginine metabolism for any mammalian species. It has become clear that a more complete understanding of arginine metabolism will require integration of information obtained from multiple approaches, including genomics, proteomics, and metabolomics.

Background: In sickle cell disease, ischemia-reperfusion injury and intravascular hemolysis produce endothelial dysfunction and vasculopathy characterized by reduced nitric oxide and arginine bioavailability. Recent functional studies of platelets in patients with sickle cell disease reveal a basally activated state, which suggests that pathological platelet activation may contribute to sickle cell disease vasculopathy., Methods and Results: Studies were therefore undertaken to examine transcriptional signaling pathways in platelets that may be dysregulated in sickle cell disease. We demonstrate and validate in the present study the feasibility of comparative platelet transcriptome studies on clinical samples from single donors by the application of RNA amplification followed by microarray-based analysis of 54,000 probe sets. Data mining an existing microarray database, we identified 220 highly abundant genes in platelets and a subset of 72 relatively platelet-specific genes, defined by >10-fold increased expression compared with the median of other cell types in the database with amplified transcripts. The highly abundant platelet transcripts found in the present study included 82% or 70% of platelet-abundant genes identified in 2 previous gene expression studies on nonamplified mRNA from pooled or apheresis samples, respectively. On comparing the platelet gene expression profiles in 18 patients with sickle cell disease in steady state to those of 12 black control subjects, at a 3-fold cutoff and 5% false-discovery rate, we identified approximately 100 differentially expressed genes, including multiple genes involved in arginine metabolism and redox homeostasis. Further characterization of these pathways with real-time polymerase chain reaction and biochemical assays revealed increased arginase II expression and activity and decreased platelet polyamine levels., Conclusions: The present studies suggest a potential pathogenic role for platelet arginase and altered arginine and polyamine metabolism in sickle cell disease and provide a novel framework for the study of disease-specific platelet biology.

Arginine, a semiessential or conditionally essential amino acid in humans, is one of the most metabolically versatile amino acids and serves as a precursor for the synthesis of urea, nitric oxide, polyamines, proline, glutamate, creatine, and agmatine. Arginine is metabolized through a complex and highly regulated set of pathways that remain incompletely understood at both the whole-body and the cellular levels. Adding to the metabolic complexity is the fact that limited arginine availability can selectively affect the expression of specific genes, most of which are themselves involved in some aspect of arginine metabolism. This overview highlights selected aspects of arginine metabolism, including areas in which our knowledge remains fragmentary and incomplete.

Context: Sickle cell disease is characterized by a state of nitric oxide resistance and limited bioavailability of l-arginine, the substrate for nitric oxide synthesis. We hypothesized that increased arginase activity and dysregulated arginine metabolism contribute to endothelial dysfunction, pulmonary hypertension, and patient outcomes., Objective: To explore the role of arginase in sickle cell disease pathogenesis, pulmonary hypertension, and mortality., Design: Plasma amino acid levels, plasma and erythrocyte arginase activities, and pulmonary hypertension status as measured by Doppler echocardiogram were prospectively obtained in outpatients with sickle cell disease. Patients were followed up for survival up to 49 months., Setting: Urban tertiary care center and community clinics in the United States between February 2001 and March 2005., Participants: Two hundred twenty-eight patients with sickle cell disease, aged 18 to 74 years, and 36 control participants., Main Outcome Measures: Plasma amino acid levels, plasma and erythrocyte arginase activities, diagnosis of pulmonary hypertension, and mortality., Results: Plasma arginase activity was significantly elevated in patients with sickle cell disease, with highest activity found in patients with secondary pulmonary hypertension. Arginase activity correlated with the arginine-ornithine ratio, and lower ratios were associated with greater severity of pulmonary hypertension and with mortality in this population (risk ratio, 2.5; 95% confidence interval [CI], 1.2-5.2; P = .006). Global arginine bioavailability, characterized by the ratio of arginine to ornithine plus citrulline, was also strongly associated with mortality (risk ratio, 3.6; 95% CI, 1.5-8.3; P<.001). Increased plasma arginase activity was correlated with increased intravascular hemolytic rate and, to a lesser extent, with markers of inflammation and soluble adhesion molecule levels., Conclusions: These data support a novel mechanism of disease in which hemolysis contributes to reduced nitric oxide bioavailability and endothelial dysfunction via release of erythrocyte arginase, which limits arginine bioavailability, and release of erythrocyte hemoglobin, which scavenges nitric oxide. The ratios of arginine to ornithine and arginine to ornithine plus citrulline are independently associated with pulmonary hypertension and increased mortality in patients with sickle cell disease.

Arginine metabolism plays a major role in cardiovascular physiology and pathophysiology, largely via nitric oxide (NO)-dependent processes. It is becoming increasingly apparent, however, that arginine metabolic enzymes other than the NO synthases can also play important roles via both NO-dependent and -independent processes. There are three sources of arginine in vivo and at least five mammalian enzymes or enzyme families that utilize arginine as substrate. Changes in arginine availability or in production of the different end products of the various arginine metabolic pathways can have distinct and profound physiologic consequences. However, our knowledge regarding the complex interplay between these pathways at the level of the whole body, specific tissues, and even individual cells, is incomplete. This review will highlight recent findings in this area that may suggest additional avenues of investigation that will allow a fuller understanding of cardiovascular physiology in health and disease.

In mammals, L-arginine is classified as a semiessential or conditionally essential amino acid, depending on the developmental stage and health status of the individual. It can be derived from proline or glutamate, with the ultimate synthetic step catalyzed by argininosuccinate lyase. L-arginine is catabolized by arginases, nitric oxide synthases, arginine:glycine amidinotransferase, and possibly also by arginine decarboxylase, resulting ultimately in the production of urea, proline, glutamate, polyamines, nitric oxide, creatine, or agmatine. There is considerable diversity in tissue-specific and stimulus-dependent regulation of expression within this group of enzymes, and the expression of several of them can be regulated at transcriptional and translational levels by changes in the concentration of L-arginine itself. Consequently, the interplay among these enzymes in the regulation of specific aspects of arginine metabolism can be quite complex. For example, nitric oxide production can be affected by the interplay between nitric oxide synthases, arginases, and argininosuccinate synthetase. This metabolic complexity can pose challenges for analyses of arginine metabolism not only because L-arginine is a substrate for several different enzymes but also because ornithine and citrulline, key products of arginine metabolism, can each be produced by multiple enzymes. This overview highlights key features of the arginine metabolic enzymes and their interactions.

Dietary supplements of L-arginine, a substrate for nitric oxide synthases, may promote formation of nitric oxide and thus may be of clinical benefit. However, the optimal level of L-arginine supplementation is unclear. The objective of this study was to evaluate the response of healthy individuals to increasing doses of L-arginine (as free acid). Twelve healthy subjects were recruited and instructed to take L-arginine for 1-week periods at daily doses of 3, 9, 21, and 30 g. At baseline and at the end of each week, 24-hour urine and fasting blood samples were collected, and weight, diastolic blood pressure, and systolic blood pressure were recorded. Samples were analyzed for L-arginine, L-citrulline, glycine, L-lysine, L-ornithine, asymmetric dimethy L-arginine, symmetric dimethy L-arginine, glucose, insulin (serum), creatinine, cGMP (urine), and total nitrates (serum and urine). Ten subjects reported adverse side effects at initial L-arginine doses of 21 g/day (five subjects) or 30 g/day (five subjects), respectively. Blood pressure and weight did not change during the supplementation period. Of the individual biochemical measures, only L-arginine, glycine, and L-ornithine concentrations changed significantly. The mean concentration of L-arginine reached a peak during supplementation at 9 g/d; however, individuals differed markedly in their response. Availability of L-arginine, relative to that of asymmetric dimethy L-arginine, increased significantly at both 9 g/day and 21 g/day. Mean values indicate that supplementation with 9 g/day of L-arginine, a dose associated with minimal adverse side effects, is sufficient to increase circulating L-arginine concentrations. However, subjects varied widely in their responses, indicating that L-arginine supplementation needs to be tailored to individuals.

Recent studies suggest that a nitric oxide (NO) deficiency and elevated arginase activity may play a role in the pathogenesis of asthma. Although much attention has been directed toward measurements of exhaled NO in asthma, no studies to date have evaluated levels of plasma arginase or arginine, the substrate for NO production, in patients with asthma. This study, therefore, measured amino acid levels, arginase activity, and nitric oxide metabolites in the blood of patients with asthma, as well as NO in exhaled breath. Although levels of virtually all amino acids were reduced, patients with asthma exhibited a striking reduction in plasma arginine levels compared with normal control subjects without asthma (45 +/- 22 vs. 94 +/- 29 microM, p < 0.0001), and serum arginase activity was elevated (1.6 +/- 0.8 vs. 0.5 +/- 0.3 micromol/ml/hour, asthma vs. control, p < 0.0001). High arginase activity in patients with asthma may contribute to low circulating arginine levels, thereby limiting arginine bioavailability and creating a NO deficiency that induces hyperreactive airways. Addressing the alterations in arginine metabolism may result in new strategies for treatment of asthma.

Purpose of Review: Arginine metabolism has been a topic of intense interest over the past 15-20 years, primarily with regard to the role of arginine as the nitrogen donor for nitric oxide synthesis. However, other important aspects of arginine metabolism, such as arginine transport and arginine catabolism via the arginases, arginine decarboxylase or agmatinase, have been less well studied. The purpose of this review is to highlight recent studies on the urea cycle, agmatine metabolism, and the arginases., Recent Findings: Recent advances include the cloning of complementary DNA encoding agmatinase, N-acetylglutamate synthetase, and proteins involved in mitochondrial arginine transport, as well as initial investigations of their regulation and tissue-specific expression. The most exciting results of studies in this area over the past year or so have indicated new roles for the arginases in health and disease, as a result of their effects on the synthesis of nitric oxide, proline, or polyamines, or on the expression of specific genes by their ability to limit the availability of free arginine., Summary: Recent studies have led to refinements in our understanding of the urea cycle. Agmatine metabolism is still largely a mystery, although the isolation of cloned cDNA for agmatinase and possibly also arginine decarboxylase should stimulate much needed investigations in this area. The most exciting findings in the field are coming from studies indicating new roles for the arginases in various diseases.

Purpose: Recent data suggest that hydroxyurea (HU) increases the production of nitric oxide (NO), a potent vasodilator. NO is normally metabolized from l-Arginine (Arg). However, in vitro and animal experiments suggest that HU is the NO donor itself. In contrast, a recent study indicates that nitric oxide synthase (NOS) may play a role. Since adults with sickle cell disease (SCD) are Arg-deficient, Arg availability may limit the ability of HU to maximally impact NO production if an NOS mechanism is involved. The authors have previously shown that Arg supplementation alone induces a paradoxical decrease in NO metabolite (NO(x)) production., Methods: The authors studied the effects of HU and Arg supplementation on NO(x) production. HU alone or HU + Arg was administered to patients with SCD at steady state, and sequential levels of Arg, serum NO(x) and exhaled NO were followed over 4 hours., Results: After HU + Arg, all patients demonstrated a significant increase in serum NO(x) production within 2 hours. When the same patients were treated with HU alone (5.1 +/- 2 micromol/L), a mixed response occurred. NO(x) levels increased in four patients and decreased in one patient (-23.3 micromol/L)., Conclusions: While Arg alone does not increase serum NO(x) production in SCD patients at steady state, it does when given together with HU. Hence, co-administration of Arg with HU may augment the NO(x) response in SCD and improve utilization of Arg in patients at steady state.

Pulmonary hypertension is a life-threatening complication of sickle cell disease. L-Arginine is the nitrogen donor for synthesis of nitric oxide, a potent vasodilator that is deficient during times of sickle cell crisis. This deficiency may play a role in pulmonary hypertension. The enzyme arginase hydrolyzes arginine to ornithine and urea, and thus, it may compete with nitric oxide synthase, leading to decreased nitric oxide production. Nitric oxide therapy by inhalation has improved pulmonary hypertension associated with acute chest syndrome in sickle cell disease, and several studies demonstrate therapeutic benefits of arginine therapy for primary and secondary pulmonary hypertension. We sought to determine the effects of arginine therapy on pulmonary hypertension in patients with sickle cell disease. Arginase activity was also determined. Oral arginine produced a 15.2% mean reduction in estimated pulmonary artery systolic pressure (63.9 +/- 13 to 54.2 +/- 12 mm Hg, p = 0.002) after 5 days of therapy in 10 patients. Arginase activity was elevated almost twofold (p = 0.07) in patients with pulmonary hypertension and may limit arginine bioavailability. With limited treatment options and a high mortality rate for patients with sickle cell disease who develop pulmonary hypertension, arginine is a promising new therapy that warrants further investigation.

L-Arginine is the only endogenous nitrogen-containing substrate of NO synthase (NOS), and it thus governs the production of NO during nervous system development as well as in disease states such as stroke, multiple sclerosis, Parkinson's disease, and HIV dementia. The "arginine paradox" refers to the dependence of cellular NO production on exogenous L-arginine concentration despite the theoretical saturation of NOS enzymes with intracellular L-arginine. Herein, we report that decreased availability of L-arginine blocked induction of NO production in cytokine-stimulated astrocytes, owing to inhibition of inducible NOS (iNOS) protein expression. However, activity of the promoter of the iNOS gene, induction of iNOS mRNA, and stability of iNOS protein were not inhibited under these conditions. Our results indicate that inhibition of iNOS activity by arginine depletion in stimulated astrocyte cultures occurs via inhibition of translation of iNOS mRNA. After stimulation by cytokines, uptake of L-arginine negatively regulates the phosphorylation status of the eukaryotic initiation factor (eIF2 alpha), which, in turn, regulates translation of iNOS mRNA. eIF2 alpha phosphorylation correlates with phosphorylation of the mammalian homolog of yeast GCN2 eIF2 alpha kinase. As the kinase activity of GCN2 is activated by phosphorylation, these findings suggest that GCN2 activity represents a proximal step in the iNOS translational regulation by availability of l-arginine. These results provide an explanation for the arginine paradox for iNOS and define a distinct mechanism by which a substrate can regulate the activity of its associated enzyme.

It has been suggested that L-arginine availability declines with advanced age, which could contribute to the endothelial dysfunction and decreased nitric oxide (NO) production that are features of aging. L-Arginine is made in the kidney and since the aging kidney develops progressive injury there may be decreased synthesis limiting availability. In this study we investigated the impact of aging on the regulation, at the gene level, of the various enzymes that synthesize L-arginine in the kidney (argininosuccinate synthetase and argininosuccinate lyase) and citrulline, the precursor of L-arginine made in the small intestine (phosphate-dependent glutaminase, carbamyl phosphate synthetase-1 and ornithine transcarbamylase). Studies were in young (3-5 months), middle-aged (11-13 months) and old (18-22 months) male and female Sprague-Dawley rats aged under barrier conditions. The plasma, renal cortical and brain cerebellar levels of L-arginine are unchanged in the old male rat, and expression of the genes involved in renal arginine synthesis and small intestinal citrulline synthesis is unchanged or upregulated with age in both males and females. This study shows that the synthesis of L-arginine is maintained with aging despite developing kidney damage. Therefore, the reduced NO generating capacity that occurs in aging must be due to downstream changes in the NO biosynthesis pathway, such as reduced abundance of NO biosynthetic enzymes.

The urea cycle is comprised of five enzymes but also requires other enzymes and mitochondrial amino acid transporters to function fully. The complete urea cycle is expressed in liver and to a small degree also in enterocytes. However, highly regulated expression of several enzymes present in the urea cycle occurs also in many other tissues, where these enzymes are involved in synthesis of nitric oxide, polyamines, proline and glutamate. Glucagon, insulin, and glucocorticoids are major regulators of the expression of urea cycle enzymes in liver. In contrast, the "urea cycle" enzymes in nonhepatic cells are regulated by a wide range of pro- and antiinflammatory cytokines and other agents. Regulation of these enzymes is largely transcriptional in virtually all cell types. This review emphasizes recent information regarding roles and regulation of urea cycle and arginine metabolic enzymes in liver and other cell types.

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.

Arginine is the sole substrate for nitric oxide (NO) synthesis by NO synthases (NOS) and promotes the proliferation and maturation of human T-cells. Arginine is also metabolized by the enzyme arginase, producing urea and ornithine, the precursor for polyamine production. We sought to determine the molecular mechanisms regulating arginase and NOS in splenic immune cells after trauma. C3H/HeN mice underwent laparotomy as simulated moderate trauma or anesthesia alone (n = 24 per group). Six, 12, 24, or 48 h later, 6 animals from each group were sacrificed, and splenectomy was performed and plasma collected. Six separate animals had neither surgery nor anesthesia and were sacrificed to provide resting values (t = 0 h). Spleen arginase I and II and iNOS mRNA abundance, arginase I protein expression, and arginase activity were determined. Plasma NO metabolites (nitrite + nitrate) were also measured. Trauma increased spleen arginase I protein expression and activity (P = 0.01) within 12 and for at least 48 h after injury and coincided with up-regulated arginase I mRNA abundance at 24 h. Neither arginase II nor iNOS mRNA abundance in the spleen was significantly increased by trauma at 24 h. Plasma nitrite + nitrate was decreased in animals 48 h post-injury compared to anesthesia controls (P < 0.05). Trauma induces up-regulation of arginase I gene expression in splenic immune cells within 24 h of injury. Arginase II is not significantly up-regulated at that time point. Arginase I, rather than iNOS appears to be the dominant route for arginine metabolism in splenic immune cells 24 h after trauma.

Arginine is one of the most versatile amino acids in animal cells, serving as a precursor for the synthesis not only of proteins but also of nitric oxide, urea, polyamines, proline, glutamate, creatine and agmatine. Of the enzymes that catalyse rate-controlling steps in arginine synthesis and catabolism, argininosuccinate synthase, the two arginase isoenzymes, the three nitric oxide synthase isoenzymes and arginine decarboxylase have been recognized in recent years as key factors in regulating newly identified aspects of arginine metabolism. In particular, changes in the activities of argininosuccinate synthase, the arginases, the inducible isoenzyme of nitric oxide synthase and also cationic amino acid transporters play major roles in determining the metabolic fates of arginine in health and disease, and recent studies have identified complex patterns of interaction among these enzymes. There is growing interest in the potential roles of the arginase isoenzymes as regulators of the synthesis of nitric oxide, polyamines, proline and glutamate. Physiological roles and relationships between the pathways of arginine synthesis and catabolism in vivo are complex and difficult to analyse, owing to compartmentalized expression of various enzymes at both organ (e.g. liver, small intestine and kidney) and subcellular (cytosol and mitochondria) levels, as well as to changes in expression during development and in response to diet, hormones and cytokines. The ongoing development of new cell lines and animal models using cDNA clones and genes for key arginine metabolic enzymes will provide new approaches more clearly elucidating the physiological roles of these enzymes.

Hepatocytes can be stimulated to express high levels of inducible nitric oxide synthase (iNOS), which utilizes arginine for nitric oxide (NO) synthesis. Hepatocytes also synthesize and catabolize arginine, an intermediate in the urea cycle, raising the possibility that the urea pathway may provide substrate for hepatic NO synthesis. To identify the sources of arginine for iNOS, we measured the release of NO-2 + NO-3 and urea in isolated rat livers perfused in a recirculation model with a Krebs-Henseleit-bicarbonate buffer containing either no added amino acid, arginine, or precursors for urea synthesis. To induce iNOS expression, rats were injected with killed Corynebacterium parvum (C. parvum) or with endotoxin. In livers from C. parvum- and endotoxin-treated rats, we found that 1) an intracellular source of arginine exists that provides substrate to iNOS; 2) additional exogenous arginine increase NO synthesis, demonstrating that endogenous arginine is insufficient for maximal NO synthesis; and 3) an increase in the rate of endogenous arginine synthesis within the urea cycle is inefficient in increasing NO synthesis, demonstrating the independence of the two pathways in the liver.

The generation of nitric oxide (NO) is largely responsible for the intracellular killing of Trypanosoma cruzi by activated macrophages. The present study was carried out to determine whether the production of NO by activated murine macrophages cultured in physiologic levels of arginine can be augmented by increasing the availability of arginine, the substrate for NO biosynthesis. Increased exogenous arginine or citrulline resulted in a significant increase in NO production and complete clearance of the parasites by activated macrophages. As citrulline fully substituted for arginine in supporting NO production and trypanocidal activity, these results demonstrate the expression of a highly active citrulline-NO cycle in activated macrophages and that levels of arginine in plasma are limiting with respect to both NO production and trypanocidal activity in these cells. The results indicate that increasing plasma substrate levels for both arginine and NO biosynthesis may represent a means of enhancing microbicidal activity in vivo.

Nitric oxide is one of the most versatile molecules produced by mammalian cells. Its role in sepsis and inflammation has been the subject of intense investigation since its discovery as a cell product in 1987. The role of arginine in sepsis and trauma has also received considerable attention, but most of the earlier studies on arginine preceded the studies on nitric oxide and the discovery that arginine serves as the nitrogen donor for nitric oxide synthesis. This review will explore the role that nitric oxide plays in sepsis and the effects of arginine metabolism on nitric oxide synthesis.

Argininosuccinate synthetase and argininosuccinate lyase catalyze the conversion of citrulline to arginine in kidney. Immunohistochemical staining of mouse kidney sections with antibodies to these two enzymes, compared with the staining patterns of known markers for proximal tubules, demonstrated that these enzymes are localized within the proximal tubules. The relative abundance of mRNA encoding argininosuccinate synthetase and argininosuccinate lyase during fetal and postnatal development of mouse kidney was also determined. Changes in relative abundance of these mRNA in kidney are coordinate during development, paralleling the developmental profile of phosphoenolpyruvate carboxykinase mRNA, which is also expressed in proximal tubules. Although relative abundances of the mRNA are comparable in liver and kidney of adult mice, the profiles of mRNA abundance during development of these two organs are distinct. The results indicate that these enzymes and their corresponding mRNA can serve as useful markers for examining the differentiation and development of renal proximal tubules in vivo and in cultured explants.

Arginine has multiple metabolic fates and thus is one of the most versatile amino acids. Not only is it metabolically interconvertible with the amino acids proline and glutamate, but it also serves as a precursor for synthesis of protein, nitric oxide, creatine, polyamines, agmatine, and urea. These processes do not all occur within each cell but are differentially expressed according to cell type, age and developmental stage, diet, and state of health or disease. Arginine metabolism also is modulated by activities of various transporters that move arginine and its metabolites across the plasma and mitochondrial membranes. Moreover, several key enzymes in arginine metabolism are expressed as multiple isozymes whose expression can change rapidly and dramatically in response to a variety of different stimuli in health and disease. As illustrated by the questions raised in this article, we currently have an imperfect and incomplete picture of arginine metabolism for any mammalian species. It has become clear that a more complete understanding of arginine metabolism will require integration of information obtained from multiple approaches, including genomics, proteomics, and metabolomics. [ABSTRACT FROM AUTHOR]

Arginine has multiple metabolic fates and thus is one of the most versatile amino acids. Not only is it metabolically interconvertible with the amino acids proline and glutamate, but it also serves as a precursor for synthesis of protein, nitric oxide, creatine, polyamines, agmatine, and urea. These processes do not all occur within each cell but are differentially expressed according to cell type, age and developmental stage, diet, and state of health or disease. Arginine metabolism also is modulated by activities of various transporters that move arginine and its metabolites across the plasma and mitochondrial membranes. Moreover, several key enzymes in arginine metabolism are expressed as multiple isozymes whose expression can change rapidly and dramatically in response to a variety of different stimuli in health and disease. As illustrated by the questions raised in this article, we currently have an imperfect and incomplete picture of arginine metabolism for any mammalian species. It has become clear that a more complete understanding of arginine metabolism will require integration of information obtained from multiple approaches, including genomics, proteomics, and metabolomics. [ABSTRACT FROM AUTHOR]

L-Arginine is the only endogenous nitrogen-containing substrate of NO synthase (NOS), and it thus governs the production of NO during nervous system development as well as in disease states such as stroke, multiple sclerosis, Parkinson's disease, and HIV dementia. The "arginine paradox" refers to the dependence of cellular NO production on exogenous L-arginine concentration despite the theoretical saturation of NOS enzymes with intracellular L-arginine. Herein, we report that decreased availability of L-arginine blocked induction of NO production in cytokine-stimulated astrocytes, owing to inhibition of inducible NOS (iNOS) protein expression. However, activity of the promoter of the iNOS gene, induction of iNOS mRNA, and stability of iNOS protein were not inhibited under these conditions. Our results indicate that inhibition of iNOS activity by arginine depletion in stimulated astrocyte cultures occurs via inhibition of translation of iNOS mRNA. After stimulation by cytokines, uptake of L-arginine negatively regulates the phosphorylation status of the eukaryotic initiation factor (elF2α), which, in turn, regulates translation of iNOS mRNA. elF2α phosphorylation correlates with phosphorylation of the mammalian homolog of yeast GCN2 elF2α kinase. As the kinase activity of GCN2 is activated by phosphorylation, these findings suggest that GCN2 activity represents a proximal step in the iNOS translational regulation by availability of L-arginine. These results provide an explanation for the arginine paradox for iNOS and define a distinct mechanism by which a substrate can regulate the activity of its associated enzyme. [ABSTRACT FROM AUTHOR]

Presents a study that examined the role of arginase I and arginase II in endothelial cell proliferation. Effect of elevated arginase expression on cell proliferation; Impact of DL-alpha-difluoromethylornithine on cell proliferation; Analysis of cellular polyamine concentration.

Examines the impact of elevated arginase I expression on cell proliferation of rat aortic smooth muscle. Precursor for polyamines production; Mechanism involving increased production of polyamines; Effect of the inhibitors on the production of urea and polyamines.

Discusses the role nitric oxide (NO) plays in sepsis and the potential impact of arginine metabolism on NO synthesis. Distribution and induction of inducible nitric oxide synthase (iNOS); Regulation of iNOS gene expression; Regulation of enzyme activity; Regulation of substrate and cofactor synthesis; Functional aspects of iNOS expression.

Summarizes the discussion on basic aspects of arginine metabolism. Significance of regulating arginine availability; Relationship between protein turnover and aspects of arginine metabolism; Mechanisms by which arginine could affect nitric oxide synthesis.

Focuses on the most salient issues that have been discussed in the symposium on arginine. Characterization of the key enzymes of arginine metabolism from a biochemist's perspective; Functional interrelationships for only some of the enzymes of arginine metabolism; Results of whole-animal studies of arginine metabolism that revealed significant variations among different species, mouse strains and gender; Effects of arginine administration in different forms of renal disease.

Summarizes the discussion on physiology of arginine metabolism. Comparison of nutritional objectives between human and livestock; Determinants of arginine deficiency in humans; Relationship between arginine metabolism and immune cells.

Increased arginase activity is implicated in cardiovascular pathogenesis. Therefore we studied systemic changes in arginase and arginine metabolism in a model of atherosclerosis. ApoE-/- and C57 mice were placed on 45 or 60% fat diets for 2 months. Results showed increased circulating arginase activity with no difference between 45% (14-fold) and 60% (16-fold) fat diet in the apoE-/-. There was a mild increase in the C57 with 45% fat (2.4-fold) and a large increase due to 60% (19-fold). In the plasma, circulating arginine was reduced in the apoE-/- and showed a decreasing trend in the C57. The plasma arginine/ornithine ratio was significantly decreased in all groups that showed profound increased plasma arginase activity. Markers of tissue (LDH) and liver (ALT) injury were markedly elevated in mice with increased arginase activity. Interestingly, blood cell gene expression in the apoE-/- fed 60% fat showed an 11-fold increase in arginase II (AII) and no increase in arginase I (AI). Tissue gene expression showed increased AI (20-5.5-fold) and AII (14- 19-fold) in aorta and heart respectively in 60% fat apoE-/- (45% to be completed) with no increase in C57. In conclusion, increased circulating arginase activity and reduced arginine are likely the result of liver and/or tissue injury. Increased arginase gene expression in the blood and tissue (apoE-/- only) are likely associated with the development of atherosclerosis. [ABSTRACT FROM AUTHOR]

Induction of arginase I (ARGI) plays a role in many inflammatory diseases. Thus, elucidation of the mechanisms involved in ARGI induction may identify targets for therapeutic intervention. Inducers of ARGI include interleukin-4 (IL-4) and agents that elevate cAMP. IL-4 and cAMP each induce ARGI in macrophages, but together they synergize to dramatically induce ARGI. The objective of this study was to determine the basis for the synergism. Immunohistochemistry showed that the degree of ARGI induction reflected differences in numbers of ARGI-expressing cells rather than in the degree of ARGI expression per cell. Previous work showed that IL-4 induction required binding of the transcription factors STAT6 and C/EBPβ to the IL-4 response element of the ARGI gene. We used chromatin immunoprecipitation (ChIP) to test the hypothesis that synergistic induction involves coordinate binding of both transcription factors to the IL-4 response element. ChIP analysis showed increased binding of both STAT6 and C/EBPβ in response to IL-4 alone, increased binding only of C/EBPβ in response to cAMP alone, but greatly increased binding of both factors in response to IL-4 + cAMP. These results indicate that synergistic induction of ARGI transcription is a stochastic process that involves a major increase in coordinate binding of both transcription factors to the IL-4 response element of the ARGI gene. [ABSTRACT FROM AUTHOR]

Introduces the symposium proceedings on arginine, held in Bermuda on April 5-6, 2004, published in the October 2004 issue of "The Journal of Nutrition."