Designing modulators of dimethylarginine dimethylaminohydrolase (DDAH): A focus on selectivity over arginase

DDAH inhibition presents a novel promising pharmaceutical strategy to lower NO formation. To date, several potent DDAH inhibitors have been published, most of them representing analogues of l-arginine. While inhibitory effects on NOSs have already been considered, selectivity over arginase has been neglected so far. In our view, the latter selectivity is more important since an additional inhibition of arginase decreases the desired effects on NO levels. Thus, we particularly focus on selectivity over arginase. We present a comprehensive selectivity profile of known DDAH inhibitors by covering their inhibitory potency on arginase. Among the studied compounds, N^ω-(2-methoxyethyl)-l-arginine (2a, L-257) that is already selective over NOSs also only modestly affected arginase activity and is thus far the most suitable DDAH inhibitor for pharmacological studies.     

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.     

Comparison of the inhibitory effect of isothiourea and mercapto-alkylguanidine derivatives on the alternative pathways of arginine metabolism in macrophages

Novel, non-arginine based compounds have been identified as potent inhibitors of nitric oxide synthase (NOS). Members of the isothiourea and mercapto-alkylguanidine classes have generated much interest, as some members of these classes show selectivity towards the inducible isoform of NOS (iNOS), which plays a role in inflammation and shock. Here we compared the effect of a number of these compounds as well as L-arginine based NOS inhibitor reference compounds on macrophage-derived and liver arginase and macrophage iNOS activities. From the nonarginine based NOS inhibitors studied only S-aminoethyl-isothiourea (AETU) caused a slight inhibition of arginase activity. This inhibition was kinetically competitive and due to the rearrangement of AETU to mercapto-ethylguanidine (MEG). The weak inhibitory effect of non-arginine based iNOS inhibitors on arginase activity further supports the view that such compounds may be of practical use for inhibition of NO production in cells simultaneously expressing iNOS and arginase.     

Alterations of NOS, arginase, and DDAH protein expression in rabbit cavernous tissue after administration of cigarette smoke extract

Cigarette smoking is an independent risk factor for vasculogenic erectile dysfunction (ED). Nitric oxide (NO) has been demonstrated to be the principal mediator of cavernous smooth muscle relaxation and penile erection. Therefore, we examined whether or not enzyme activities and factors involved in the NO generation pathway are affected in rabbit corpus cavernosum after administration of nicotine- and tar-free cigarette smoke extract (CSE). CSE was prepared by bubbling a stream of cigarette smoke into phosphate-buffered saline. CSE was injected subcutaneously into adult male rabbits once a day for 5 wk. In the CSE group, significantly decreased cyclic GMP production as a marker of NO generation was associated with attenuated overall nitric oxide synthase (NOS) activity, enhanced arginase activity, accumulation of endogenous NOS inhibitors such as monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), and decreased dimethylarginine dimethylaminohydrolase (DDAH) activity as an metabolizing enzyme of endogenous NOS inhibitors. Neuronal NOS (nNOS) and DDAH I protein expression were decreased without altering endothelial NOS expression, while arginase I expression was upregulated. These results suggest that impaired NO production would result from blunted NOS activity, which is possibly brought about by the downregulation of nNOS protein, accumulation of endogenous NOS inhibitors, and enhanced arginase activity together with upregulation of arginase I protein in cavernous tissue. The impaired DDAH activity due to decreased expression of DDAH I protein would result in an accumulation of endogenous NOS inhibitors with CSE. These alterations may be relevant to induction of the erectile dysfunction following CSE.     

Piceatannol is more effective than resveratrol in restoring endothelial cell dimethylarginine dimethylaminohydrolase expression and activity after high-glucose oxidative stress

Glucose-induced oxidative stress is involved in endothelial dysfunction. Dimethylarginine dimethylaminohydrolase (DDAH) and arginase are regulators of the endothelial NO synthase (eNOS). This study aimed to compare the effect of two polyphenolic antioxidants, resveratrol and piceatannol, on DDAH and arginase pathways in bovine aortic endothelial cells under 25 mM glucose for 24 h. DDAH activity and expression were decreased in these cells as compared to control cells, whereas arginase activity was unchanged. DDAH inhibition led to intracellular accumulation of asymmetric dimethylarginine (ADMA), a natural inhibitor of eNOS. Under these conditions, cell pre-treatment with resveratrol (0.1-10 μM) restored basal DDAH activity and ADMA level with a dose-dependent effect. Piceatannol acted as resveratrol on DDAH pathway but at 10-fold lower concentrations. Resveratrol and piceatannol restored DDAH activity even in the presence of splitomicin, a specific inhibitor of Sirtuin 1. These results suggest potential therapeutic intervention targeting resveratrol or piceatannol administration to improve endothelial dysfunction.     

Esomeprazole covalently interacts with the cardiovascular enzyme dimethylarginine dimethylaminohydrolase: Insights into the cardiovascular risk of proton pump inhibitors

BackgroundProton pump inhibitors (PPIs) are widely prescribed drugs for the treatment of gastroesophageal reflux disease (GERD). Several meta-analysis studies have reported associations between prolonged use of PPIs and major adverse cardiovascular events. However, interaction of PPIs with biological molecules involved in cardiovascular health is incompletely characterized. Dimethylarginine dimethylaminohydrolase (DDAH) is a cardiovascular enzyme expressed in cardiomyocytes, and other somatic cell types in one of two isotypes (DDAH1 and DDAH2) to metabolize asymmetric dimethylarginine (ADMA); a cardiovascular risk factor and competitive inhibitor of nitric oxide synthases (NOSs).MethodsWe performed high throughput drug screening of over 130,000 small molecules to discover human DDAH1 inhibitors and found that PPIs directly inhibit DDAH1. We expressed and purified the enzyme for structural and mass spectrometry proteomics studies to understand how a prototype PPI, esomeprazole, interacts with DDAH1. We also performed molecular docking studies to model the interaction of DDAH1 with esomeprazole. X-ray crystallography was used to determine the structure of DDAH1 alone and bound to esomeprazole at resolutions ranging from 1.6 to 2.9 Å.ResultsAnalysis of the enzyme active site shows that esomeprazole interacts with the active site cysteine (Cys273) of DDAH1. The structural studies were corroborated by mass spectrometry which indicated that cysteine was targeted by esomeprazole to inactivate DDAH1.ConclusionsThe inhibition of this important cardiovascular enzyme by a PPI may help explain the reported association of PPI use and increased cardiovascular risk in patients and the general population.General significanceOur study calls for pharmacovigilance studies to monitor adverse cardiovascular events in chronic PPI users.     

Role of DDAH-1 in lipid peroxidation product-mediated inhibition of endothelial NO generation

Altered nitric oxide (NO) biosynthesis is thought to play a role in the initiation and progression of atherosclerosis and may contribute to increased risk seen in other cardiovascular diseases. It is hypothesized that altered NO bioavailability may result from an increase in endogenous NO synthase (NOS) inhibitors, asymmetric dimethly araginine (ADMA), and N(G)-monomethyl arginine, which are normally metabolized by dimethyarginine dimethylamine hydrolase (DDAH). Lipid hydroperoxides and their degradation products are generated during inflammation and oxidative stress and have been implicated in the pathogenesis of cardiovascular disorders. Here, we show that the lipid hydroperoxide degradation product 4-hydroxy-2-nonenal (4-HNE) causes a dose-dependent decrease in NO generation from bovine aortic endothelial cells, accompanied by a decrease in DDAH enzyme activity. The inhibitory effects of 4-HNE (50 microM) on endothelial NO production were partially reversed with L-Arg supplementation (1 mM). Overexpression of human DDAH-1 along with antioxidant supplementation completely restored endothelial NO production following exposure to 4-HNE (50 microM). These results demonstrate a critical role for the endogenous methylarginines in the pathogenesis of endothelial dysfunction. Because lipid hydroperoxides and their degradation products are known to be involved in atherosclerosis, modulation of DDAH and methylarginines may serve as a novel therapeutic target in the treatment of cardiovascular disorders associated with oxidative stress.     

Paradoxical effect of l-arginine: Acceleration of endothelial cell senescence

We have recently shown that inhibition of nitric oxide (NO) synthesis by asymmetrical dimethylarginine (ADMA) accelerated endothelial cell (EC) senescence which was prevented by coincubation with l-arginine; however the effect of long-term treatment of l-arginine alone on senescence of ECs have not been investigated. Human ECs were cultured in medium containing different concentrations of l-arginine until senescence. l-Arginine paradoxically accelerated senescence indicated by inhibiting telomerase activity. Moreover, l-arginine decreased NO metabolites, increased peroxynitrite, and 8-iso-prostaglandin F_2α formation. In old cells, the mRNA expression of human amino acid transporter (hCAT)2B, the activity and protein expression of arginase II were upregulated indicated by enhanced urea, l-ornithine, and l-arginine consumption. Inhibition of arginase activity, or transfection with arginase II siRNA prevented l-arginine-accelerated senescence. The most possible explanation for the paradoxical acceleration of senescence by l-arginine so far may be the translational and posttranslational activation of arginase II.     

Endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox

L-Arginine, the substrate of nitric oxide (NO) synthases (NOSs), is found in the mammalian organism at concentrations by far exceeding K(M) values of these enzymes. Therefore, additional L-arginine should not enhance NO formation. In vivo, however, increasing L-arginine concentration in plasma has been shown repeatedly to increase NO production. This phenomenon has been named the L-arginine paradox; it has found no satisfactory explanation so far. In the present work, evidence for the hypothesis that the endogenous NOS inhibitors methylarginines, asymmetric dimethylarginine being the most powerful (IC(50) 1.5 microM), are responsible for the L-arginine paradox is presented.     

Modulation of nitric oxide (NO) biosynthesis in lactobacilli

We characterized effects of nitric oxide synthase (NOS) substrate L-arginine and classical inhibitors of mammalian NOS on nitric oxide (NO) biosynthesis in probiotic bacteria Lactobacillus plantarum 8P-A3. NO-synthase origin of nitric oxide detected by fluorescent NO indicator 1,2-diaminoanthraquinone (DAA) was confirmed by induction of NO production by exogenous L-arginine. None of the used inhibitors of three isoforms of mammalian NOSs (L-NAME, L-NIL, nNOS inhibitor I) showed significant inhibitory effect of lactobacillar NO-synthase activity.     

Dimethylarginine dimethylaminohydrolase-2 overexpression improves impaired nitric oxide synthesis of endothelial cells induced by glycated protein.

Nitric oxide (NO) synthesis is modulated by dimethylarginine dimethylaminohydrolase (DDAH) via metabolizing asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor. This study investigated whether glycosylated bovine serum albumin (GBSA) could impair NO synthesis by inhibition of DDAH expression and activity, and whether DDAH2 overexpression could reverse the impaired NO synthesis induced by GBSA in endothelial cells. Overexpression of DDAH2 gene was established by liposome-mediated gene transfection in ECV304 endothelial cell line. Cells were incubated with 1.70 mmol/L GBSA for 48h. And the expressions of DDAH1 and DDAH2, gene activities of DDAH and NOS in cells, as well as concentrations of ADMA and NO in media were assayed. The activity of DDAH and expression of DDAH2 gene but not DDAH1 gene were inhibited in endothelial cells after exposure to GBSA, whereas the concentrations of ADMA were increased concomitantly with the decrease of NOS activity in cells and NO production in media. Overexpression of DDAH2 gene could prevent the inhibition of DDAH activity induced by GBSA (0.55+/-0.02 vs 0.42+/-0.02U/g pro; n=3; P<0.05), decrease ADMA concentration (0.59+/-0.04 vs 1.13+/-0.11 micromol/L; n=3; P<0.01), and increase NOS activity and NO production (53.77+/-3.40 vs 34.59+/-2.57 micromol/L; P<0.05) compared with untransfected cells treated with GBSA. These results suggest that decreased DDAH activity and subsequent elevated endogenous ADMA are implicated in the inhibition of NO synthesis induced by GBSA, and overexpression of DDAH2 gene can prevent these changes in DDAH/ADMA/NOS/NO pathway of endothelial cells exposed to GBSA.     

Efficient formation of nitric oxide from selective oxidation of N-aryl N'-hydroxyguanidines by inducible nitric oxide synthase

Inducible nitric oxide synthase (NOS II) efficiently catalyzes the oxidation of N-(4-chlorophenyl)N'-hydroxyguanidine 1 by NADPH and O2, with concomitant formation of the corresponding urea and NO. The characteristics of this reaction are very similar to those of the NOS-dependent oxidation of endogenous Nomega-hydroxy-L-arginine (NOHA), i.e., (i) the formation of products resulting from an oxidation of the substrate C=N(OH) bond, the corresponding urea and NO, in a 1:1 molar ratio, (ii) the absolute requirement of the tetrahydrobiopterin (BH4) cofactor for NO formation, and (iii) the strong inhibitory effects of L-arginine (L-arg) and classical inhibitors of NOSs. N-Hydroxyguanidine 1 is not as good a substrate for NOS II as is NOHA (Km = 500 microM versus 15 microM for NOHA). However, it leads to relatively high rates of NO formation which are only 4-fold lower than those obtained with NOHA (Vm = 390 +/- 50 nmol NO min-1 mg protein-1, corresponding roughly to 100 turnovers min-1). Preliminary results indicate that some other N-aryl N'-hydroxyguanidines exhibit a similar behavior. These results show for the first time that simple exogenous compounds may act as NO donors after oxidative activation by NOSs. They also suggest a possible implication of NOSs in the oxidative metabolism of certain classes of xenobiotics.     

Rational design of novel irreversible inhibitors for human arginase

Parasites have developed a variety of strategies for invading hosts and escaping their immune response. A common mechanism by which parasites escape nitric oxide (NO) toxicity is the activation of host arginase. This activation leads to a depletion of l-arginine, which is the substrate for NO synthase, resulting in lower levels of NO and increased production of polyamines that are necessary for parasite growth and differentiation. For this reason, small molecule inhibitors for arginase show promise as new anti-parasitic chemotherapeutics. However, few arginase inhibitors have been reported. Here, we describe the discovery of novel irreversible arginase inhibitors, and their characterization using biochemical, kinetic, and structural studies. Importantly, we determined the site on human arginase that is labeled by one of the small molecule inhibitors. The tandem mass spectra data show that the inhibitor occupies the enzyme active site and forms a covalent bond with Thr135 of arginase. These findings pave the way for the development of more potent and selective irreversible arginase inhibitors.     

The DDAH/ADMA pathway is a critical regulator of NO signalling in vascular homeostasis

NO is an important regulator of cardiovascular remodelling and function. ADMA, an endogenous L-arginine analogue, reduces NO production by inhibiting the activity of NOS. ADMA levels in turn, are regulated by DDAH, which metabolises ADMA. High levels of ADMA and dysregulated DDAH activity are risk factors for cardiovascular disease and morbidity. To investigate this link, the DDAH I null mouse has been recently generated and has a lethal phenotype. Studies on vascular function in the DDAH I heterozygous knockout mouse, which is viable, demonstrates a causal link between reduced DDAH I activity, increased ADMA levels and reduced NO signalling and vascular dysfunction. In another study, detailed in vitro analyses reveal that the DDAH/ADMA pathway critically regulates endothelial cell motility and angiogenesis and establishes some of the molecular mechanisms involved. These studies highlight the importance of DDAH and ADMA in regulating NO dependent vascular homeostasis.Key words: asymmetric dimethylarginine (ADMA), dimethylarginine dimethylaminohydrolase (DDAH), nitric oxide (NO), angiogenesis, endothelial, motilityNO is generated from L-arginine by NOS; a process which is competitively inhibited by the arginine analogues ADMA and L-NMMA. These endogenous factors are products of proteolytic degradation of methylated proteins. ADMA and L-NMMA are metabolised by DDAH I and II, thereby enhancing NO generation. Of relevance to vascular biology, dysfunctional DDAH activity and ADMA accumulation are risk factors for cardiovascular disorders, including hypertension, artherosclerosis, diabetes, insulin resistance, hypercholesterolemia and homocysteinemia (reviewed in ref. ¹).The DDAH I null mouse was generated recently by Leiper et al.² to facilitate investigation of the role of the DDAH/ADMA pathway in the pathology of cardiovascular disorders. While the absence of DDAH I causes a lethal phenotype, heterozygotes (HT) did not display any obvious abnormalities. However, ADMA levels were raised in tissues and plasma, in association with raised blood pressure and systemic vascular resistance, and reduced cardiac output and heart rate. Synthetic DDAH I inhibitors were designed by the authors and were shown by crystallography to bind to the active site of the enzyme and induce local distortions at this region. Confirming that loss of DDAH I was responsible for ADMA accumulation, these inhibitors enhanced ADMA levels in wildtype mice, and resulted in cardiovascular changes similar to those seen in the HT background. Inhibitor treatment also promoted ADMA release from wildtype blood vessels maintained ex vivo, indicating that the DDAH/ADMA pathway is directly responsible for maintaining cardiovascular function in this model.Evidence was also presented for a causal link between ADMA metabolism and reduced NO levels. In an ex vivo model, aortic rings from HT mice displayed enhanced phenylephrine-induced contraction and reduced acetylcholine-induced relaxation, while DDAH I inhibitors induced similar responses in aortic rings from wildtype mice; indicative of reduced levels of endothelial-derived NO. Further demonstrating an ADMA/NO-dependent mechanism, exogenous L-arginine restored a normal response to these vasomodulators in the HT model (by competing with ADMA for interaction with NOS). Similarly, cultured endothelial cells from HT vessels produced more ADMA and less NO than cells from wildtype vessels, and DDAH I inhibitors induced a similar phenotype in wildtype endothelial cells. The significance of DDAH I/ADMA and NO in vascular disease was tested in a disease model. Endotoxic shock was induced in rats by intravenous infusion of LPS, which induces excess NO production, resulting in systemic hypotension. After blood pressure had fallen by 20%, infusion of a DDAH I inhibitor was able to rapidly stabilise blood pressure, in accordance with inhibition of NO production through reduced ADMA metabolism. Thus, when DDAH I is reduced, ADMA is increased and endogenous NO inhibited, resulting in altered vascular function.Another related study investigated a mechanistic understanding of the role of ADMA/DDAH/NO in angiogenesis.³ The authors demonstrated that ADMA regulates endothelial cell motility and phenotype by inhibiting NO-dependent changes in activity of Rho-GTPases; key mediators of cytoskeletal dynamics and motility. Treatment of pulmonary artery endothelial cells with ADMA enhanced stress fibres and focal adhesion formation in conjunction with increased activity of RhoA in pull-down assays. In accordance with these observations, motility, tracked by time-lapse microscopy, was inhibited by ADMA treatment, and ADMA effects were reversed by a Rho kinase inhibitor (Y-27632) or by adenoviral-mediated gene transfer of a dominant negative RhoA mutant. RhoA activity is mediated by PKG, which mediates RhoA-Ser188 phosphorylation, preventing RhoA localization to the membrane and inhibiting its activity.⁴ In further support of a RhoA-dependent mechanism, ADMA reduced phosphorylation at RhoA-Ser188, while a PKG activator was also able to revert ADMA effects on motility. Further, a non-phosphorylatable mutant of RhoA, Ala188RhoA, or a specific PKG inhibitor, each inhibited cell motility to a similar level as ADMA treatment alone. Inhibition of NO production and endothelial cell motility by ADMA was also reversed by a NO donor, SNAP, or by DDAH I or II overexpression via adenovirus-mediated gene transfer. Thus, reduction of NO/PKG levels by ADMA reduces RhoA phosphorylation at Ser188 resulting in enhancement of RhoA activity and inhibition of cell motility.The significance of these molecular mechanisms to angiogenesis was demonstrated using endothelial cells and aortic ring explants from HT DDAH I and wildtype mice. HT endothelial cells, which secrete more ADMA and produce less NO than their wildtype counterparts, exhibit enhanced RhoA activity and stress fibre formation in conjunction with reduced motility. Reduced sprouting from ex vivo aortic rings was also observed in the HT model, which was mimicked by addition of exogenous ADMA in the wildtype background. These data demonstrate that in vivo, DDAH/ADMA levels are likely to play a key role in control of endothelial cell motility and angiogenesis by regulating NO production.     

The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and p38 mitogen-activated protein kinase in IL-13-mediated arginase I induction in macrophages: its implications in IL-13-inhibited nitric oxide production

In macrophages, L-arginine can be used by NO synthase and arginase to form NO and urea, respectively. Therefore, activation of arginase may be an effective mechanism for regulating NO production in macrophages through substrate competition. Here, we examined whether IL-13 up-regulates arginase and thus reduces NO production from LPS-activated macrophages. The signaling molecules involved in IL-13-induced arginase activation were also determined. Results showed that IL-13 increased arginase activity through de novo synthesis of the arginase I mRNA and protein. The activation of arginase was preceded by a transient increase in intracellular cAMP, tyrosine kinase phosphorylation, and p38 mitogen-activated protein kinase (MAPK) activation. Exogenous cAMP also increased arginase activity and enhanced the effect of IL-13 on arginase induction. The induction of arginase was abolished by a protein kinase A (PKA) inhibitor, KT5720, and was down-regulated by tyrosine kinase inhibitors and a p38 MAPK inhibitor, SB203580. However, inhibition of p38 MAPK had no effect on either the IL-13-increased intracellular cAMP or the exogenous cAMP-induced arginase activation, suggesting that p38 MAPK signaling is parallel to the cAMP/PKA pathway. Furthermore, the induction of arginase was insensitive to the protein kinase C and p44/p42 MAPK kinase inhibitors. Finally, IL-13 significantly inhibited NO production from LPS-activated macrophages, and this effect was reversed by an arginase inhibitor, L-norvaline. Together, these data demonstrate for the first time that IL-13 down-regulates NO production through arginase induction via cAMP/PKA, tyrosine kinase, and p38 MAPK signalings and underline the importance of arginase in the immunosuppressive activity of IL-13 in activated macrophages.     

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.     

N(delta)-Methylated L-arginine derivatives and their effects on the nitric oxide generating system

So far N(delta)-methyl-l-arginine (MA) is only detected in yeast cells. Assuming that MA also exists in mammalians we examined possible physiological effects of N(delta)-methylated l-arginine derivatives on the nitric oxide generating system, that is, nitric oxide synthase (NOS), arginase and dimethylarginine dimethylaminohydrolase (DDAH). N(delta)-methyl-l-citrulline (MC) turned out to be a weak non-specific inhibitor of nitric oxide synthases. Moreover, MA is hydroxylated by all human NOS isoforms to N(omega)-hydroxy-N(delta)-methyl-l-arginine (NHAM) but not converted further. This hydroxylated intermediate, however, was detected to be a potent inhibitor of bovine liver arginase with a K(i) of 17.1+/-2.2 microM.     

Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells

Elevated levels of serum uric acid (UA) are commonly associated with primary pulmonary hypertension but have generally not been thought to have any causal role. Recent experimental studies, however, have suggested that UA may affect various vasoactive mediators. We therefore tested the hypothesis that UA might alter nitric oxide (NO) levels in pulmonary arterial endothelial cells (PAEC). In isolated porcine pulmonary artery segments (PAS), UA (7.5 mg/dl) inhibits acetylcholine-induced vasodilation. The incubation of PAEC with UA caused a dose-dependent decrease in NO and cGMP production stimulated by bradykinin or Ca(2+)-ionophore A23187. We explored cellular mechanisms by which UA might cause reduced NO production focusing on the effects of UA on the l-arginine-endothelial NO synthase (eNOS) and l-arginine-arginase pathways. Incubation of PAEC with different concentrations of UA (2.5-15 mg/dl) for 24 h did not affect l-[(3)H]arginine uptake or activity/expression of eNOS. However, PAEC incubated with UA (7.5 mg/dl; 24 h) released more urea in culture media than control PAEC, suggesting that arginase activation might be involved in the UA effect. Kinetic analysis of arginase activity in PAEC lysates and rat liver and kidney homogenates demonstrated that UA activated arginase by increasing its affinity for l-arginine. An inhibitor of arginase (S)-(2-boronoethyl)-l-cysteine prevented UA-induced reduction of A23187-stimulated cGMP production by PAEC and abolished UA-induced inhibition of acetylcholine-stimulated vasodilation in PAS. We conclude that UA-induced arginase activation is a potential mechanism for reduction of NO production in PAEC.