Diabetes-induced vascular dysfunction involves arginase I

Arginase can cause vascular dysfunction by competing with nitric oxide synthase for l-arginine and by increasing cell proliferation and collagen formation, which promote vascular fibrosis/stiffening. We have shown that increased arginase expression/activity contribute to vascular endothelial cell (EC) dysfunction. Here, we examined the roles of the two arginase isoforms, arginase I and II (AI and AII, respectively), in this process. Experiments were performed using streptozotocin-induced diabetic mice: wild-type (WT) mice and knockout mice lacking the AII isoform alone (AI(+/+)AII(-/-)) or in combination with partial deletion of AI (AI(+/-)AII (-/-)). EC-dependent vasorelaxation of aortic rings and arterial fibrosis and stiffness were assessed in relation to arginase activity and expression. Diabetes reduced mean EC-dependent vasorelaxation markedly in diabetic WT and AI(+/+)AII(-/-) aortas (53% and 44% vs. controls, respectively) compared with a 27% decrease in AI(+/-)AII (-/-) vessels. Coronary fibrosis was also increased in diabetic WT and AI(+/+)AII(-/-) mice (1.9- and 1.7-fold vs. controls, respectively) but was not altered in AI(+/-)AII (-/-) diabetic mice. Carotid stiffness was increased by 142% in WT diabetic mice compared with 51% in AI(+/+)AII(-/-) mice and 19% in AI(+/-)AII (-/-) mice. In diabetic WT and AI(+/+)AII(-/-) mice, aortic arginase activity and AI expression were significantly increased compared with control mice, but neither parameter was altered in AI(+/-)AII (-/-) mice. In summary, AI(+/-)AII (-/-) mice exhibit better EC-dependent vasodilation and less vascular stiffness and coronary fibrosis compared with diabetic WT and AI(+/+)AII(-/-) mice. These data indicate a major involvement of AI in diabetes-induced vascular dysfunction.     

Arginase expression and modulation of IL-1β-induced nitric oxide generation in rat and human islets of Langerhans

Proinflammatory cytokine induction of NO synthesis may contribute to the destruction of pancreatic beta cells leading to type 1 diabetes. The NO synthase substrate arginine can also be metabolized to ornithine and urea in a reaction catalyzed by cytosolic (AI) or mitochondrial (AII) isoforms of arginase. Recent evidence suggests that the rate of NO generation is dependent on the relative activities of NO synthase and arginase. The objectives of this study were (i) to identify the arginase isoforms expressed in rat and human islets of Langerhans and a rat beta cell line, RINm5F and (ii) to investigate the competition for arginine between NO synthase and arginase in IL-1β-treated rat islets. Arginase activity was detected in rat islets (fresh tissue, 346 mU/mg protein; cultured, 587 mU/mg), cultured human islets (56 mU/mg), RINm5F cells (376 mU/mg), rat kidney (238 mU/mg), and rat liver (6119 mU/mg). Using Western blots, AI was shown to be the predominant isoform expressed in rat islets and in RINm5F cells while human islets expressed far more AII than AI. Rat islets were cultured in medium containing 1.14, 0.1, and 0.01 mM arginine and treated with IL-1β and the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH). IL-1β-induced NO generation was unaffected by ABH at 1.14 mM arginine, but significantly increased at 0.1 and 0.01 mM arginine. These findings suggest that the level of islet arginase activity can regulate the rate of induced NO generation and this may be relevant to the insulitis process leading to beta cell destruction in type 1 diabetes.     

Arginase expression and modulation of IL-1beta-induced nitric oxide generation in rat and human islets of Langerhans.

Proinflammatory cytokine induction of NO synthesis may contribute to the destruction of pancreatic beta cells leading to type 1 diabetes. The NO synthase substrate arginine can also be metabolized to ornithine and urea in a reaction catalyzed by cytosolic (AI) or mitochondrial (AII) isoforms of arginase. Recent evidence suggests that the rate of NO generation is dependent on the relative activities of NO synthase and arginase. The objectives of this study were (i). to identify the arginase isoforms expressed in rat and human islets of Langerhans and a rat beta cell line, RINm5F and (ii). to investigate the competition for arginine between NO synthase and arginase in IL-1beta-treated rat islets. Arginase activity was detected in rat islets (fresh tissue, 346 mU/mg protein; cultured, 587 mU/mg), cultured human islets (56 mU/mg), RINm5F cells (376 mU/mg), rat kidney (238 mU/mg), and rat liver (6119 mU/mg). Using Western blots, AI was shown to be the predominant isoform expressed in rat islets and in RINm5F cells while human islets expressed far more AII than AI. Rat islets were cultured in medium containing 1.14, 0.1, and 0.01 mM arginine and treated with IL-1beta and the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH). IL-1beta-induced NO generation was unaffected by ABH at 1.14 mM arginine, but significantly increased at 0.1 and 0.01 mM arginine. These findings suggest that the level of islet arginase activity can regulate the rate of induced NO generation and this may be relevant to the insulitis process leading to beta cell destruction in type 1 diabetes.     

Inhibition of arginase ameliorates experimental ulcerative colitis in mice

Abstract

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

Nitric oxide inhibition of ERK1/2 activity in cells expressing neuronal nitric-oxide synthase

Neuronal nitric-oxide synthase (nNOS) is a constitutively expressed enzyme responsible for the production of nitric oxide (NO*) from l-arginine and O2. Nitric oxide is an intra- and intercellular messenger that mediates a diversity of signaling pathways in target cells. In the absence of l-arginine, nNOS has been shown to generate superoxide (O2*). Superoxide, either directly or through its self-dismutation to H2O2, is likewise believed to be a cell-signaling agent. Because nNOS can generate NO* and O2*, we examined the activation of cellular signal transduction pathways in nNOS-transfected cells grown in the presence or absence of l-arginine. Spin trapping/EPR spectroscopy confirmed that stimulated nNOS-transfected cells grown in an l-arginine environment secreted NO* into the surrounding milieu. Production of NO* blocked Ca2+ ionophore-induced activation of the ERK1/2 through a mechanism involving inhibition of the Ras G-protein and Raf-1 kinase. In contrast, ERK activation was largely unaffected in nNOS-transfected cells grown in l-arginine-free media. Inhibition of nNOS-generated NO* with the competitive NOS inhibitor, NG-nitro-l-arginine methyl ester, in cells grown in l-arginine restored ERK1/2 activation to levels similar to that found when nNOS was activated in l-arginine-free media. These findings indicate that nNOS can differentially regulate the ERK signal transduction pathway in a manner dependent on the presence of l-arginine and the production of NO*.     

Neuronal nitric oxide synthase: expression in rat parietal cells

Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.     

Effects of the new arginase inhibitor N(omega)-hydroxy-nor-L-arginine on NO synthase activity in murine macrophages.

In stimulated murine macrophage, arginase and nitric oxide synthase (NOS) compete for their common substrate, l-arginine. The objectives of this study were (i) to test the new alpha-amino acid N(omega)-hydroxy-nor-l-arginine (nor-NOHA) as a new selective arginase inhibitor and (ii) to elucidate the effects of arginase inhibition on l-arginine utilization by an inducible NOS. Nor-NOHA is about 40-fold more potent than N(omega)-hydroxy-l-arginine (NOHA), an intermediate in the l-arginine/NO pathway, to inhibit the hydrolysis of l-arginine to l-ornithine catalyzed by unstimulated murine macrophages (IC(50) values 12 +/- 5 and 400 +/- 50 microM, respectively). Stimulation of murine macrophages with interferon-gamma and lipopolysaccharide (IFN-gamma + LPS) results in clear expression of an inducible NOS (iNOS) and to an increase in arginase activity. Nor-NOHA is also a potent inhibitor of arginase in IFN-gamma + LPS-stimulated macrophage (IC(50) value 10 +/- 3 microM). In contrast to NOHA, nor-NOHA is neither a substrate nor an inhibitor for iNOS and it appears as a useful tool to study the interplays between arginase and NOS. Inhibition of arginase by nor-NOHA increases nitrite and l-citrulline accumulation for incubation times higher than 12 h, under our conditions. Our results allow the determination of the kinetic parameters of the two competitive pathways and the proposal of a simple model which readily explains the differences observed between experiments. This model readily accounts for the observed effects and should be useful to predict the consequences of arginase inhibition in the presence of an active NOS on l-arginine availability.     

Seasonality in expression and distribution of nitric oxide synthase isoforms in the testis of the catfish, Clarias batrachus: Role of nitric oxide in testosterone production

Nitric oxide (NO) is a well-recognized versatile signaling molecule. It is produced by catalytic action of nitric oxide synthase (NOS) on L-arginine in a variety of animal tissues. Existence of different isoforms of NOS has been shown in mammalian testis, but report on their presence in the testis of ectothermic vertebrates is non-existent. This study demonstrates the differential expressions of two isoforms of nitric oxide synthase (neuronal-nNOS and inducible-iNOS) like molecules in different cell types in the testis of seasonally breeding catfish, Clarias batrachus through immunohistochemistry. Positive immunoprecipitation of nNOS and iNOS like molecules were detected in germ cells as well as interstitial cells only in the recrudescing and fully mature fish. The immunoreactions differed in intensity and varied with changing reproductive status. Treatment of adult male fish with NO donor, sodium nitroprusside, and a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME) increased and decreased the total nitrate and nitrite concentration in the testis, respectively. Sodium nitroprusside and L-NAME also induced simultaneous decline and rise in the testicular testosterone level, respectively. These findings, thus, suggest that NOS isoforms are expressed variedly in different cell types in the testis of reproductively active fish. This investigation also suggests that NO inhibits testosterone production in the testis.     

Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells

The neuronal and endothelial isoforms of nitric oxide (NO) synthase (nNOS and eNOS, respectively) both catalyze the production of NO but are regulated differently. Stably transfected HEK 293 cell lines containing nNOS, eNOS, and a soluble mutant of eNOS were therefore established to compare their activity in a common cellular environment. NOS activity was determined by measuring L-[3H]citrulline production in homogenates and intact cells, the conversion of oxyhemoglobin to methemoglobin, and the production of cGMP. The results indicate that nNOS is more active than eNOS, both in unstimulated as well as calcium-stimulated cells. Under basal conditions, the soluble mutant of eNOS appeared to be slightly more active than wild-type eNOS in terms of NO and cGMP formation, suggesting that membrane association may be crucial for inhibition of basal NO release but is not required for stimulation by Ca2+-mobilizing agents. The maximal activity of soluble guanylate cyclase was significantly reduced by transfection with wild-type eNOS due to downregulation of mRNA expression. These results demonstrate that nNOS and eNOS behave differently even in an identical cellular environment.     

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.     

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.     

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.     

Gene transfer with inducible nitric oxide synthase decreases production of urea by arginase in pulmonary arterial endothelial cells

Nitric oxide (NO) is a vasodilator produced from L-arginine (L-Arg) by NO synthase (NOS). Gene therapy for hypertensive disorders has been proposed using the inducible isoform of NOS (iNOS). L-Arg also can be metabolized to urea and L-ornithine (L-Orn) by arginase, and L-Orn can be metabolized to proline and/or polyamines, which are vital for cellular proliferation. To determine the effect of iNOS gene transfer on arginase, we transfected bovine pulmonary arterial endothelial cells (bPAEC) with an adenoviral vector containing the gene for iNOS (AdiNOS). As expected, NO production in AdiNOS bPAEC was substantially greater than in control bPAEC. Although urea production was significantly less in the AdiNOS bPAEC than in the control bPAEC, despite similar levels of arginase I protein, AdiNOS transfection of bPAEC had no effect on the uptake of L-Arg. Inhibiting NO production with Nomega-nitro-L-arginine methyl ester increased urea production, and inhibiting urea production with L-valine increased nitrite production, in AdiNOS bPAEC. The addition of L-Arg to the medium increased urea production by AdiNOS bPAEC in a concentration-dependent manner. Thus, in these iNOS-transfected bPAEC, the transfected iNOS and native arginase compete for a common intracellular pool of L-Arg. This competition for substrate resulted in impaired proliferation in the AdiNOS-transfected bPAEC. These findings suggest that the use of iNOS gene therapy for pulmonary hypertensive disorders may not only be beneficial through NO-mediated pulmonary vasodilation but also may decrease vascular remodeling by limiting L-Orn production by native arginase.     

Arginase II Downregulates Nitric Oxide (NO) Production and Prevents NO-mediated Apoptosis in Murine Macrophage-derived RAW 264.7 Cells

Excess nitric oxide (NO) induces apoptosis of some cell types, including macrophages. As NO is synthesized by NO synthase (NOS) from arginine, a common substrate of arginase, these two enzymes compete for arginine. There are two known isoforms of arginase, types I and II. Using murine macrophage-like RAW 264.7 cells, we asked if the induction of arginase II would downregulate NO production and hence prevent apoptosis. When cells were exposed to lipopolysaccharide (LPS) and interferon-γ (IFN-γ), the inducible form of NOS (iNOS) was induced, production of NO was elevated, and apoptosis followed. When dexamethasone and cAMP were further added, both iNOS and arginase II were induced, NO production was much decreased, and apoptosis was prevented. When the cells were transfected with an arginase II expression plasmid and treated with LPS/IFN-γ, some cells were rescued from apoptosis. An arginase I expression plasmid was also effective. On the other hand, transfection with the arginase II plasmid did not prevent apoptosis when a NO donor SNAP or a high concentration (12 mM) of arginine was added. These results indicate that arginase II prevents NO-dependent apoptosis of RAW 264.7 cells by depleting intracellular arginine and by decreasing NO production.     

Arginine metabolic pathways involved in the modulation of tumor-induced angiogenesis by macrophages

Neovascularization, an essential step for tumor progression and metastasis development, can be modulated by the presence of macrophages (Mps) in the tumor microenvironment. The ability of Mps to regulate the angiogenicity of the LMM3 tumor cell line was studied. Peritoneal Mps from LMM3 tumor-bearing mice (TMps) potentiate in vivo LMM3 angiogenicity. These results were confirmed by CD31 immunoblotting assays. The activity of TMps depended on nitric oxide synthase (NOS) and arginase (A) activity. By immunoblotting we evidenced that AI and AII isoforms were up-regulated in TMps while the inducible and neuronal NOS isoforms were highly expressed in normal Mps. TMps might positively modulate tumor growth by stimulating angiogenic cascade mainly through polyamine synthesis.     

Regulatory role of arginase I and II in nitric oxide, polyamine, and proline syntheses in endothelial cells

Endothelial cells (EC) metabolize L-arginine mainly by arginase, which exists as two distinct isoforms, arginase I and II. To understand the roles of arginase isoforms in EC arginine metabolism, bovine coronary venular EC were stably transfected with the Escherichia coli lacZ gene (lacZ-EC, control), rat arginase I cDNA (AI-EC), or mouse arginase II cDNA (AII-EC). Western blots and enzymatic assays confirmed high-level expression of arginase I in the cytosol of AI-EC and of arginase II in mitochondria of AII-EC. For determining arginine catabolism, EC were cultured for 24 h in DMEM containing 0.4 mM L-arginine plus [1-(14)C]arginine. Urea formation, which accounted for nearly all arginine consumption by these cells, was enhanced by 616 and 157% in AI-EC and AII-EC, respectively, compared with lacZ-EC. Arginine uptake was 31-33% greater in AI-EC and AII-EC than in lacZ-EC. Intracellular arginine content was 25 and 11% lower in AI-EC and AII-EC, respectively, compared with lacZ-EC. Basal nitric oxide (NO) production was reduced by 60% in AI-EC and by 47% in AII-EC. Glutamate and proline production from arginine increased by 164 and 928% in AI-EC and by 79 and 295% in AII-EC, respectively, compared with lacZ-EC. Intracellular content of putrescine and spermidine was increased by 275 and 53% in AI-EC and by 158 and 43% in AII-EC, respectively, compared with lacZ-EC. Our results indicate that arginase expression can modulate NO synthesis in bovine venular EC and that basal levels of arginase I and II are limiting for endothelial syntheses of polyamines, proline, and glutamate and may have important implications for wound healing, angiogenesis, and cardiovascular function.     

Endothelial cell nitric oxide inhibits aldosterone synthesis in zona glomerulosa cells: modulation by oxygen

The regulation of aldosterone synthesis by endogenous nitric oxide (NO) was examined in cultured cells of the adrenal cortex. Endothelial NO synthase (eNOS) was detected by Western blot in cultured adrenal endothelial cells (ECs) but not in zona glomerulosa (ZG) cells or adrenal fibroblasts. Neither inducible (iNOS) nor neuronal NOS (nNOS) isoforms were detected in the cells. Only ECs had NOS activity and converted [(3)H]L-arginine to [(3)H]L-citrulline. Angiotensin II (ANG II, 100 nM) increased EC production of nitrate/nitrite by 2.4-fold. Coincubation with ECs or treatment with DETA nonoate increased the fluorescence of ZG cells loaded with an NO-sensitive dye, diaminofluorescein 2 diacetate (DAF-2 DA). DETA nonoate inhibited ANG II (1 nM) and potassium (10 mM) -stimulated aldosterone release in a concentration-related manner. This inhibitory effect of NO was enhanced >10-fold by decreasing the oxygen concentration from 21 to 8%. Coincubation of EC and ZG cells in 8% oxygen inhibited ANG II-induced aldosterone release, and inhibition was reversed by blockade of NOS. These findings indicate that adrenal EC-derived NO inhibits aldosterone release by cultured ZG cells and that the sensitivity to NO inhibition is increased at low oxygen concentrations.     

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.     

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.     

Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation

Background

Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO) production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using guinea pig tracheal open-ring preparations, we now investigated the involvement of arginase in the modulation of neuronal nitric oxide synthase (nNOS)-mediated relaxation induced by inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation.

Methods

Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz)-induced relaxation was measured in tracheal preparations precontracted to 30% with histamine, in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to the EFS-induced relaxation was assessed by the nonselective NOS inhibitor L-NNA (0.1 mM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor nor-NOHA (10 μM). Furthermore, the role of substrate availability to nNOS in EFS-induced relaxation was measured in the presence of various concentrations of exogenous L-arginine.

Results

EFS induced a frequency-dependent relaxation, ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz, which was inhibited with the NOS inhibitor L-NNA by 78.0 ± 10.5% at 0.5 Hz to 26.7 ± 7.7% at 8 Hz (P < 0.01 all). In contrast, the arginase inhibitor nor-NOHA increased EFS-induced relaxation by 3.3 ± 1.2-fold at 0.5 Hz to 1.2 ± 0.1-fold at 4 Hz (P < 0.05 all), which was reversed by L-NNA to the level of control airways in the presence of L-NNA (P < 0.01 all). Similar to nor-NOHA, exogenous L-arginine increased EFS-induced airway relaxation (P < 0.05 all).

Conclusion

The results indicate that endogenous arginase activity attenuates iNANC nerve-mediated airway relaxation by inhibition of NO generation, presumably by limiting L-arginine availability to nNOS.