L-arginine Metabolism Research Articles

Interferon-Gamma-Induced Nitric Oxide Inhibits the Proliferation of Murine Renal Cell Carcinoma Cells

Renal cell carcinoma (RCC) remains one of the most resistant tumors to systemic chemotherapy, radiotherapy, and immunotherapy. Despite great progress in understanding the basic biology of RCC, the rate of responses in animal models and clinical trials using interferons (IFNs) has not improved significantly. It is likely that the lack of responses can be due to the tumor's ability to develop tumor escape strategies. Currently, the use of targeted therapies has improved the clinical outcomes of patients with RCC and is associated with an increase of Th1-cytokine responses (IFNγ), indicating the importance of IFNγ in inhibiting tumor proliferation. Thus, the present study was designed to investigate a new mechanism by which IFNγ mediates direct anti-proliferative effects against murine renal cell carcinoma cell lines. When cultured RCC cell lines were exposed to murine recombinant IFNγ, a dose dependent growth inhibition in CL-2 and CL-19 cells was observed; this effect was not observed in Renca cells. Growth inhibition in CL-2 and CL-19 cell lines was associated with the intracellular induction of nitric oxide synthase (iNOS) protein, resulting in a sustained elevation of nitric oxide (NO) and citrulline, and a decrease in arginase activity. The inhibition of cell proliferation appears to be due to an arrest in the cell cycle. The results indicate that in certain RCC cell lines, IFNγ modulates L-arginine metabolism by shifting from arginase to iNOS activity, thereby developing a potent inhibitory mechanism to encumber tumor cell proliferation and survival. Elucidating the cellular events triggered by IFNγ in murine RCC cell lines will permit anti-tumor effects to be exploited in the development of new combination therapies that interfere with L-arginine metabolism to effectively combat RCC in patients.

L-Arginine Metabolism in the Lung: Reciprocal Regulation of the NOS and Arginase Pathways

It has been known for 20 years that the production of nitric oxide (NO) by the NO synthase (NOS) isozymes is important in the maintenance of airways tone. Over the past decade, however, it has become increasingly apparent that competition between the NOS and arginase pathways for L-arginine, limits NO production. Imbalances between these pathways have been implicated in the airways hyperresponsiveness (AHR) of asthma. Thus, a delicate balance between the NOS and arginase pathways is maintained through the intracellular synthesis of endogenous NOS and arginase inhibitors. More recently, the liberation of methylarginines has emerged as an additional modifier of L-arginine uptake and metabolism in the lung. In this review we discuss the reciprocal regulation of the NOS and arginase pathways and methylarginines and their roles in the airways hyperresponsiveness of asthma.

Citrulline: A potential immunomodulator in sepsis

Sepsis leads to a complex systemic response of cytokines (both pro- and anti-inflammatory) and more recently recognized adipokine mediators. Endothelial nitric oxide (NO) may be a key component in regulating this response, but the pharmacologic manipulation of endothelial NO via L-arginine supplementation or inhibitors has provided inconsistent clinical data related to outcomes. These failures are related to the metabolism of L-arginine in the liver, toxicity of L-arginine, and asymmetric dimethylarginine inhibition, all of which may explain the "arginine paradox." L-citrulline (CIT) offers a potentially valuable means of supplementing arginine and therefore impacting favorably NO availability. The goal of this study was to determine whether CIT supplementation altered the systemic response of mediators and cytokines in a rat model of sepsis with varying degrees of severity. Sepsis was induced with 2 models of cecal ligation and puncture (CLP) of varying severity in Wistar rats. CIT supplementation was provided to half the animals as 8% CIT-supplemented feed for 3 weeks. Baseline mediator levels were assessed in the Wistar rats followed by comparison of the following groups at days 0, 1, and 3: sham-operated; CLP 8-mm (localized); and CLP 12-mm (extensive). The following analyses were performed in the groups: interleukin-6 (IL-6), IL-8, IL-10, resistin, and adiponectin levels (enzyme-linked immunosorbent assay performed in duplicate). L-arginine and CIT were measured with high-performance liquid chromatography combined with mass spectrometry. Ninety-eight Wistar rats were evaluated, and survival was similar in both sepsis models with and without CIT. Serum IL-6 levels were lower in the CIT/CLP 8-mm group compared to the standard rat chow (STD)/CLP 8-mm group (41 vs 117 pg/mL; P = .011) on postoperative day 3. Serum IL-8 and IL-10 responses were similar across all groups. Serum resistin levels were lower in the CIT/CLP 12-mm group compared to the STD/CLP 12-mm group in the more severe sepsis model on day 3 (19 vs 38 ng/mL; P < .0001). The levels of serum L-arginine were greater in the CIT-supplemented animals compared to STD rodent diet animals before surgical insult (86.3 vs 294.0 μM; P = .004). Adiponectin was not affected by CIT supplementation. CIT may decrease the proinflammatory mediator response (IL-6 and resistin) without impairing the secretion of anti-inflammatory mediators (IL-10 and adiponectin) and thereby provide a safe means of immunomodulation that preserves the anti-inflammatory mediator response.

Hypoxia-reduced nitric oxide synthase activity is partially explained by higher arginase-2 activity and cellular redistribution in human umbilical vein endothelium

Hypoxia relates with altered placental vasodilation, and in isolated endothelial cells, it reduces activity of the endothelial nitric oxide synthase (eNOS) and l-arginine transport. It has been reported that arginase-2 expression, an alternative pathway for l-arginine metabolism, is increased in adult endothelial cells exposed to hypoxia as well as in pre-eclamptic placentae. We studied in human umbilical vein endothelial cells (HUVEC) whether hypoxia-reduced NO synthesis results from increased arginase-mediated l-arginine metabolism and changes in subcellular localization of eNOS and arginase-2. In HUVEC exposed (24 h) to 5% (normoxia) or 2% (hypoxia) oxygen, l-arginine transport kinetics, arginase activity (urea assay), and NO synthase (NOS) activity (l-citrulline assay) were determined. Arginase-1, arginase-2 and eNOS expression were determined by RT-PCR and Western blot. Subcellular localization of arginase-2 and eNOS were studied using confocal microscopy and indirect immunofluorescence. Experiments were done in absence or presence of S-(2-boronoethyl)-l-cysteine-HCl (BEC, arginase inhibitor) or NG-nitro-l-arginine methyl ester (l-NAME). Hypoxia-induced reduction in eNOS activity was associated with a reduction in eNOS phosphorylation at Serine-1177 and increased phosphorylation at Threonine-495. This was paralleled with an induction in arginase-2 expression and activity, and decreased l-arginine transport. In hypoxia the arginase inhibition, restored NO synthesis and l-arginine transport, without changes in the eNOS post-translational modification status. Hypoxia increased arginase-2/eNOS colocalization, and eNOS redistribution to the cell periphery. Altogether these data reinforce the thought that eNOS cell location, post-translational modification and substrate availability are important mechanisms regulating eNOS activity. If these mechanisms occur in pregnancy diseases where feto-placental oxygen levels are reduced remains to be clarified.

Effect of Lactobacillus plantarum and l-arginine against endotoxin-induced liver injury in a rat model

Aims Use of probiotics, alone or as adjunct to other established therapies, has been reported to have potential benefits. Recently, we have reported protective potential of probiotic against Salmonella-induced liver injury. However, co-supplementation with prebiotics did not result in meaningful synergism at systemic level. Owing to the action of probiotics at the mucosal level and of arginine at systemic level, the present study was designed to evaluate the effect of Lactobacillus plantarum alone or in conjunction with arginine to combat endotoxin-mediated liver injury in rats. Main methods Bacterial endotoxin/lipopolysaccharide (LPS) was injected intraperitoneally and animals were sacrificed 8 h post-challenge. Efficacy of L. plantarum alone or in conjunction with l-arginine was determined on the basis of enzyme markers, histology, levels of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) in addition to identification of amino acids by paper chromatography. Key findings Prior supplementation of LPS-challenged rats with L. plantarum (10 10 CFU per rat given orally for 10 days) demonstrated decreased levels of liver enzymes, NO and TNF-α. Interestingly, complementing Lactobacillus with arginine revealed a synergistic decrease not only in the liver markers but also in NO and TNF-α along with increased intensity of ornithine and methionine. Histological evidence also confirmed the protective efficacy of probiotic in conjunction with l-arginine. Significance Presence of ornithine and methionine in the probiotic-arginine co-supplemented group suggests involvement of arginase-induced synthesis of polyamines. This study highlights that L. plantarum may direct l-arginine metabolism towards polyamine synthesis thereby exhibiting synergistic effect against liver injury.

Scopolamine impairs behavioural function and arginine metabolism in the rat dentate gyrus

Alzheimer’s disease (AD) is a neurodegenerative disorder with progressive memory loss. It has been shown that the cholinergic neurotransmission deficit is one of the neurochemical characteristics of AD, and that l-arginine and its metabolites also play a prominent role in AD pathogenesis. Scopolamine, a non-selective muscarinic receptor antagonist, blocks cholinergic neurotransmission and impairs behavioural function, including learning and memory. This study investigated the effects of scopolamine on animals’ behavioural performance and l-arginine metabolism in the hippocampus and prefrontal cortex. Rats were given intraperitoneal injections of scopolamine (0.8 mg/kg) or saline (1 ml/kg) and tested in the Y-maze, open field, water maze and elevated plus maze 30 min post-treatment. After completion of the behavioural testing, the CA1, CA2/3 and dentate gyrus (DG) sub-regions of the hippocampus and the prefrontal cortex were harvested to measure the activity and protein expression of nitric oxide synthase (NOS) and arginase, and the levels of l-arginine, l-citrulline, l-ornithine, agmatine, putrescine, spermidine, spermine, glutamate and GABA. Scopolamine treated rats displayed reduced alternation and exploratory behaviour, increased swimming speed and impaired spatial learning and memory. There were significantly decreased NOS activity, increased arginase activity, and increased l-ornithine and putrescine levels in the DG, but not other regions examined, in the scopolamine treated rats as compared to the controls. These findings suggest that scopolamine impairs behavioural function and alters l-arginine metabolism in the DG sub-region of the hippocampus specifically. The underlying mechanisms of it remain to be explored further.

Melatonin treatment following stroke induction modulates l-arginine metabolism

The efficacy of melatonin treatment in experimental stroke has been established. Some of the neuroprotective properties have been attributed to its anti-oxidant and anti-inflammatory effects. Nitric oxide synthases (NOS) and cyclooxygenases (COX) are considered to have a significant role in the inflammatory milieu occurring in acute stroke. While previous reports have shown that pretreatment with melatonin in a stroke model can modulate NOS isoforms, the effect of post-treatment with melatonin on l-arginine metabolism has not been investigated. This study initially examined the effect of melatonin (1 nm-1 mm) on l-arginine metabolism pathways in human fibrosarcoma fibroblasts (HT-1080) fibroblasts. Evidence of neuroprotection with melatonin was evaluated in rats subjected to middle cerebral artery occlusion (MCAO). Animals were treated with three daily doses of 5 mg/kg i.p., starting 1 hr after the onset of ischemia. Constitutive NOS activity but not expression was significantly increased by in vitro exposure (72 hr) to melatonin. In addition, melatonin treatment increased arginase activity by increasing arginase II expression. In vivo studies showed that melatonin treatment after MCAO significantly inhibited inducible NOS activity and attenuated expression of the inducible isoform, resulting in decreased total NOS activity and tissue nitrite levels. COX activity was significantly reduced with melatonin treatment. The neuroprotective anti-inflammatory effects of melatonin were consistent with the substantial reduction in infarct volume throughout the cortex and striatum and recovery of mitochondrial enzyme activities. The evidence presented here suggests that modulation of l-arginine metabolism by melatonin make it a valuable neuroprotective therapy for stroke.

Arginase and arginine dysregulation in asthma.

In recent years, evidence has accumulated indicating that the enzyme arginase, which converts L-arginine into L-ornithine and urea, plays a key role in the pathogenesis of pulmonary disorders such as asthma through dysregulation of L-arginine metabolism and modulation of nitric oxide (NO) homeostasis. Allergic asthma is characterized by airway hyperresponsiveness, inflammation, and remodeling. Through substrate competition, arginase decreases bioavailability of L-arginine for nitric oxide synthase (NOS), thereby limiting NO production with subsequent effects on airway tone and inflammation. By decreasing L-arginine bioavailability, arginase may also contribute to the uncoupling of NOS and the formation of the proinflammatory oxidant peroxynitrite in the airways. Finally, arginase may play a role in the development of chronic airway remodeling through formation of L-ornithine with downstream production of polyamines and L-proline, which are involved in processes of cellular proliferation and collagen deposition. Further research on modulation of arginase activity and L-arginine bioavailability may reveal promising novel therapeutic strategies for asthma.

ANTARCTIC FISH METABOLIC RESPONSES AS POTENTIAL BIOMARKERS OF ENVIRONMENTAL IMPACT

The principal objective of this review is to discuss the energy metabolism, of L-arginine, antioxidant defense and the xenobiotics of Antarctic fishes as possible markers of environmental change. With the establishment of the circumpolar current and the physical isolation of the region, Antarctic fishes evolved under stable low temperatures. The elevated endemism, low diversity, and the probable stenthermability motivated a number of studies on Antarctic fishes. The presence of enzymes with physical and chemical properties adaptaded to low temperatures was evident in various studies and fortified the cold adaptation concept of these organisms. By ratifying the Antarctica Environmental Protection Treaty (Madrid protocol), the member countries committed themselves to monitoring the environment of the region. Though, it is one of the most pristine regions of the planet, increasing human activity in Antarctica has given rise to concerns about pollution from combustion oils, heavy metals and sewage. As the energy sustainability of the tissues requires that the velocity of the ATP generator systems should be in step with the energy demand, changes in the environment would lead to elevation in the energy demand of the organisms, which in turn demanded an adjustment of the ATP synthesis velocity. In these cases the enzymatic upregulation and downregulation have been used as biomarkers of the energy metabolism. Traditionally, the antioxidant defense enzymes and the xenobiotic metabolism of fishes have been used as biomarkers to access the natural or anthropic environmental change. However, these studies must be correlated the levels of enzymes with the principal risk factors in the environmental pollution. L-arginine metabolism enzymes have been used as biomarkers of the physiological response in mammals, but there are few studies on Antarctic fishes. The central role of the amino acid L-arginine in the synthesis of nitric oxide, polyamines and as a signal in various physiological processes has drawn the attention of scientists. The tissue distribution and the isoforms distribution, along with its control via upregulation and downregulation of the arginase enzymes, nitric oxide synthase and ornithina decarboxylase in Antarctic fishes are potential biomarkers for environmental change.

L-Arginine Supplementation and Metabolism in Asthma

l-Arginine, the amino acid substrate for nitric oxide synthase, has been tested as a therapeutic intervention in a variety of chronic diseases and is commonly used as a nutritional supplement. In this study, we hypothesized that a subset of moderate to severe persistent asthma patients would benefit from supplementation with l-arginine by transiently increasing nitric oxide levels, resulting in bronchodilation and a reduction in inflammation. The pilot study consisted of a 3 month randomized, double-blind, placebo-controlled trial of l-arginine (0.05 g/kg twice daily) in patients with moderate to severe asthma. We measured spirometry, exhaled breath nitric oxide, serum arginine metabolites, questionnaire scores, daily medication use and PEFR with the primary endpoint being the number of minor exacerbations at three months. Interim analysis of the 20 subjects showed no difference in the number of exacerbations, exhaled nitric oxide levels or lung function between groups, though participants in the l-arginine group had higher serum l-arginine at day 60 (2.0 ± 0.6 × 10−3 vs. 1.1 ± 0.2 × 10−3 μmol/L, p < 0.05), ornithine at day 30 (2.4 ± 0.9 vs. 1.2 ± 0.3 μmol/L serum, p < 0.05) and ADMA at day 30 (6.0 ± 1.5 × 10−1 vs. 2.6 ± 0.6 × 10−1 μmol/L serum, p < 0.05) on average compared to the placebo group. The study was terminated prematurely. Supplementing asthma subjects with l-arginine increases plasma levels; whether subgroups might benefit from such supplementation requires further study.

Review: Differential placental macrovascular and microvascular endothelial dysfunction in gestational diabetes

Human endothelial dysfunction is a common feature in many diseases of pregnancy, such as gestational diabetes (GD). Metabolic changes include abnormal synthesis of nitric oxide (NO) and abnormal membrane transport of l-arginine and adenosine in primary cultures of human umbilical vein (HUVEC, macrovascular) and placental microvillus (hPMEC, microvascular) endothelial cells. These alterations are associated with modifications in the expression and activity of endothelial (eNOS) and inducible (iNOS) NO synthases, respectively, an effect that is maintained at least up to passage 5 in culture. HUVEC and hPMEC exhibit expression and activity of the human cationic amino acid transporter 1 (hCAT-1), equilibrative nucleoside transporters 1 (hENT1) and hENT2, as well as the corresponding SLC7A1, SLC29A1 and SLC29A2 gene promoter activities. Altered gene expression results from increased NO level, protein kinase C, mitogen-activated protein kinases, and hCHOP-C/EBPα transcription factor activation. Reduced ENT-mediated adenosine transport in GD is associated with stimulation of the l-arginine/NO pathway, and mainly due to reduced expression and activity of hENT1. In addition, hENT2 activity seems able to restore the reduced adenosine transport in GD. Additionally, insulin exerts a differential modulation of endothelial cells from macrocirculation compared with microcirculation, possibly due to expression of different insulin receptor isoforms. It is suggested that a common functional characteristic leading to changes in the bioavailability of adenosine and metabolism of l-arginine is evidenced by human fetal micro and macrovascular endothelium in GD.

Age-related and blood pressure-independent reduction in aortic stiffness after kidney transplantation

Aortic stiffness is a novel cardiovascular risk factor in patients with chronic kidney disease (CKD). The purpose of the present study is to examine whether there is a blood pressure-independent improvement in aortic stiffness 3 months after successful kidney transplantation (KTx), and whether this improvement is age-dependent. In this prospective, longitudinal observational study, we studied hemodynamic and biological parameters prior to and 3 months after a KTx in 52 stage 5 CKD patients. Aortic stiffness was measured by carotido-femoral pulse wave velocity (c-f PWV) and enhanced central wave reflection was evaluated by the heart rate-adjusted central augmentation index (AIx) by means of arterial tonometry. Endothelin-1, L-arginine, asymmetric dimethylarginine (biomarkers of endothelial dysfunction), pentosidine (advanced glycation end-products) and mineral metabolism parameters were also measured. After adjusting for the reduction in mean blood pressure, c-f PWV decreased significantly from 12.1 ± 3.3 to 11.6 ± 2.3 m/s (P < 0.05). In an analysis stratified by age, this improvement was only present in patients older than 50 years of age as compared with patients younger than 50 years of age (-5.5 ± 2.2 vs. 2.1 ± 1.9%, P < 0.05). AIx decreased from 22 ± 11 to 14 ± 13% (P < 0.01), but this reduction was not age-dependent. We also observed a similar degree of improvement in the biomarker levels of endothelial dysfunction and pentosidine in both groups. This study shows for the first time that there is an age-dependent improvement in aortic stiffness after KTx. These observations suggest that older patients may have an added cardiovascular risk reduction after a successful KTx.

Arginase and YKL-40, Effectors of Immunosuppressive Myeloid Cells, Are Over-Expressed In the Bone Marrow of Most Chronic Myelomonocytic Leukemia Patients, and Are Potential Prognostic Biomarkers In Myelodysplastic Syndrome

AbstractAbstract 1855 Introduction:Solid tumors commonly recruit and condition immature myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMΦs), which impair immune-surveillance and facilitate growth. Whether they play a pathogenic role in hematological malignancies is not known. Our previous studies in an MDS/CMML model (SHIP-/- mice) revealed MΦs that are skewed to an alternatively activated (M2) phenotype (reminiscent of TAMΦs), characterized by arginase 1 (Arg1)-mediated redirection of L-arginine metabolism away from cytotoxic NO and towards the production of healing intermediates, as well as over-expression of the Th2-skewing and anti-apoptotic chitinase-like protein, Ym1. Moreover, MDSCs are also increased in SHIP-/- mice, leading to T-cell suppression. We therefore hypothesized that MDSCs/M2-MΦs may be expanded in the BM of MDS and CMML patients and/or their properties hijacked by the malignant clones. Methods:With informed consent, BM mononuclear cells (MNCs) and plasma were obtained from 40 specimens in the Sunnybrook MDS Biobank (6 normals, 11 CMML-1, 1 CMML-2, 1 RARS, 2 del(5q), 6 RCMD, 2 RCMD-RS, 8 RAEB-1, and 3 RAEB-2) (20 at presentation; 20 at follow-up). BM plasma was subjected to YKL-40 ELISA and Quantibody (RayBiotech) cytokine array (bFGF, BMP6, IL-1α, IL-1β, IL-6, PDGF-BB, TGFβ1, TNFα, VEGF, and VEGF-R2). BM-MNC protein lysates were prepared and subjected to arginase enzymatic assay, and Arg1/YKL-40 Westerns. Micro-vessel density (MVD) was recorded as the concordant counts of two observers per mm2 of H&E BM biopsies. Surrogate MVD analysis was performed by automated CD31 immunohistochemistry (IHC) analysis (Vias, Ventana). Linear regression and unpaired, two-tailed t tests were performed with Prism (GraphPad) software. Results:Mean BM-MNC arginase activity (μg urea/h/μg protein, recorded as % of control mean) and BM plasma YKL-40 levels were significantly higher in CMML patients, as compared to controls (350 ± 69 % vs 100 ± 5 %, p = 0.022, and 300 ± 32 μg/L vs 150 ± 22 μg/L, p = 0.0033, respectively). Westerns confirmed intracellular Arg1/YKL-40 over-expression in CMML BM-MNCs. Heterogeneous Arg1/YKL-40 over-expression was observed in MDS patients, and was strongly associated with the lowest risk IPSS and WPSS MDS scoring categories (Figure 1) (CMML not included). Moreover, high Arg1, but not YKL-40, was able to further discriminate IPSS Low/Int1-risk patients with poor prognosis using the low-risk MD Anderson score (Figure 1). This low-risk MDS/high-Arg1/YKL-40 profile was confirmed by negative correlations with (poor-prognostic) MVD (Arg1: p = 0.049, YKL-40: p = 0.089) (similar trends with CD31), albeit in a seemingly paradoxical pro-angiogenic BM plasma milieu (trends to increased bFGF, PDGF-BB, VEGF, and VEGF-R2). These results were unexpected, given that Arg1/YKL-40 are normally thought to be pro-angiogenic, and were reminiscent of recent studies suggesting VEGF-mediated recruitment of suppressive myeloid cells may inhibit angiogenesis and tumor progression in some situations (Stockmann et al., Nature 456:814-8). Accordingly, MDS BM arginase activity was associated with a larger BM myeloid compartment (using > 60% as a cutoff; p = 0.042) and a striking linear correlation with peripheral blood (PB) neutrophil counts on the day of BM collection (p = 0.0003), likely reflecting BM and systemic expansion of suppressive myeloid populations. MDS BM-MNC Arg1/YKL-40 Westerns, stratified by PB neutrophil counts, confirmed these associations. Conclusions:1) Arg1/YKL-40, effectors of MDSCs/M2-MΦs, are over-expressed in BM MNCs and plasma in most CMML patients, as predicted from SHIP-/- mouse studies. Perturbations in SHIP and associated pathways may warrant investigation in CMML. 2) Arg1/YKL-40 are over-expressed in low-risk MDS patients, as assessed using 3 prognostic scoring systems. Their immune-suppressive nature and pro-proliferative/anti-apoptotic effects may create a permissive environment for low-risk MDS clones. 3) The unexpected association of high Arg1/YKL-40 with a pro-angiogenic BM milieu and paradoxically low MVD, may be in keeping with the ability of MDSCs/M2-MΦs to inhibit angiogenesis under certain conditions. Understanding what regulates the “immune/angiogenic switch” to low suppressive myeloid/high MVD/high-risk MDS may have important future treatment implications. [Display omitted] Disclosures:Krystal:Aquinox Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Extracorporeal photochemotherapy induces arginase 1 in patients with graft versus host disease

The benefits of extracorporeal photochemotherapy (ECP; psoralen and UVA exposure of blood mononuclear cells) in graft-versus-host-disease (GVHD) are well-recognized, but the mechanisms of action remain elusive. As the metabolism of l-arginine in immune cells is known to play a role in immune tolerance, we investigated the effect of ECP on arginine metabolism, and the influence of extracellular l -arginine concentration on the response to ECP in cells from patients on therapy by ECP for a GVHD and healthy donors cultured before and after ECP in the presence of different concentrations of arginine (0, 50, 100, 200 and 1000μmol/l). At baseline arginine was not metabolized through the same pathway in patients and donors. When cells were exposed to ECP, the production of ornithine but not NO° was enhanced, while mRNA of arginase 1 was up-regulated but not INOS. In GVHD patients, increasing arginine concentration resulted in down-regulation of IFNγ and TNFα mRNA expression, whereas IL10 was up-regulated especially at physiological plasma levels (between 0 and 100μM). Overall, our study shows that ECP orients the metabolism of arginine toward the arginase pathway together with shifting the cytokine profile toward IL-10, providing new insights into the enigmatic mechanism of action of ECP.

Androgen-Regulated Expression of Arginase 1, Arginase 2 and Interleukin-8 in Human Prostate Cancer

BackgroundProstate cancer (PCa) is the most frequently diagnosed cancer in North American men. Androgen-deprivation therapy (ADT) accentuates the infiltration of immune cells within the prostate. However, the immunosuppressive pathways regulated by androgens in PCa are not well characterized. Arginase 2 (ARG2) expression by PCa cells leads to a reduced activation of tumor-specific T cells. Our hypothesis was that androgens could regulate the expression of ARG2 by PCa cells.Methodology/Principal FindingsIn this report, we demonstrate that both ARG1 and ARG2 are expressed by hormone-sensitive (HS) and hormone-refractory (HR) PCa cell lines, with the LNCaP cells having the highest arginase activity. In prostate tissue samples, ARG2 was more expressed in normal and non-malignant prostatic tissues compared to tumor tissues. Following androgen stimulation of LNCaP cells with 10 nM R1881, both ARG1 and ARG2 were overexpressed. The regulation of arginase expression following androgen stimulation was dependent on the androgen receptor (AR), as a siRNA treatment targeting the AR inhibited both ARG1 and ARG2 overexpression. This observation was correlated in vivo in patients by immunohistochemistry. Patients treated by ADT prior to surgery had lower ARG2 expression in both non-malignant and malignant tissues. Furthermore, ARG1 and ARG2 were enzymatically active and their decreased expression by siRNA resulted in reduced overall arginase activity and l-arginine metabolism. The decreased ARG1 and ARG2 expression also translated with diminished LNCaP cells cell growth and increased PBMC activation following exposure to LNCaP cells conditioned media. Finally, we found that interleukin-8 (IL-8) was also upregulated following androgen stimulation and that it directly increased the expression of ARG1 and ARG2 in the absence of androgens.Conclusion/SignificanceOur data provides the first detailed in vitro and in vivo account of an androgen-regulated immunosuppressive pathway in human PCa through the expression of ARG1, ARG2 and IL-8.

Up‐regulating arginase contributes to amelioration of chilling stress and the antioxidant system in cherry tomato fruits

Arginase, which plays an important role in regulating the metabolism of L-arginine (Arg) in mammalian cells, has been reported to be involved in stress responses in higher plants. In view of the well-established roles of polyamines, nitric oxide and proline in plant tolerance to chilling stress, arginase may play an important role in fruit chilling tolerance by regulating the metabolism of Arg. However, the current information available on this is very limited. Cherry tomatoes (Lycopersicon esculentum cv.Messina) at the mature green stage were treated with Arg or N(ω)--hydroxy-nor-L-arginine (nor-NOHA) and analysed for chilling injury, electrolyte leakage, malondialdehyde and proline contents and arginase and antioxidant enzyme (superoxide dismutase, catalase, peroxidaseand ascorbate peroxidase) activities during cold storage. The effects of low temperature on the transcriptional levels of two arginase genes (LeARG1 and LeARG2) were also evaluated. LeARG1 and LeARG2 expression and arginase activity were significantly induced by low temperature. Compared with the control, Arg treatment alleviated fruit chilling injury, reduced electrolyte leakage, malondialdehyde content and peroxidaseactivity and increased other parameters. Treatment with nor-NOHA caused the opposite effects. Up-regulation of arginase activity and gene expression may be a chilling tolerance strategy in cherry tomato fruit. Inhibition of chilling-induced arginase activity could aggravate chilling injury and oxidation damage. Arginase appears to play an important role in the chilling resistance process of cherry tomato fruit induced by Arg.

Nitric oxide is involved in the regulation of marble-burying behavior

The aim of this study was to characterize the behavioral effect of modulators of NO synthesis in the mouse marble-burying test. We found that the non-selective NOS inhibitor 7-nitroindazole (7-NI) as well as the more selective neuronal and inducible NOS inhibitor 1-(2-trifluoromethylphenyl)imidazole (TRIM) decreased the number of marbles buried. Treatment with NO precursor l-arginine alone (500 mg/kg) had no effect in this paradigm, but counteracted the effects of paroxetine (10 mg/kg) and citalopram (10 mg/kg). Moreover, agmatine (20 and 50 mg/kg i.p), a molecule related to l-arginine metabolism, inhibited the marble-burying behavior. This effect of agmatine was not related to inhibition of NO synthesis as the drug did not decrease the nitrate and nitrite level in the brain tissue.We conclude that NOS inhibitors and agmatine dose-dependently inhibit the marble-burying behavior in mice. Inhibition of nNOS seems to play a key role in the behavioral effects of NOS inhibitors, whereas agmatine seems to have a different mechanism of action. Moreover, enhancement of NO synthesis by l-arginine reversed the effect of SSRI antidepressants, further demonstrating the role of NO in regulating the marble-burying behavior.

Symmetric allosteric mechanism of hexameric Escherichia coli arginine repressor exploits competition between L-arginine ligands and resident arginine residues.

An elegantly simple and probably ancient molecular mechanism of allostery is described for the Escherichia coli arginine repressor ArgR, the master feedback regulator of transcription in L-arginine metabolism. Molecular dynamics simulations with ArgRC, the hexameric domain that binds L-arginine with negative cooperativity, reveal that conserved arginine and aspartate residues in each ligand-binding pocket promote rotational oscillation of apoArgRC trimers by engagement and release of hydrogen-bonded salt bridges. Binding of exogenous L-arginine displaces resident arginine residues and arrests oscillation, shifting the equilibrium quaternary ensemble and promoting motions that maintain the configurational entropy of the system. A single L-arg ligand is necessary and sufficient to arrest oscillation, and enables formation of a cooperative hydrogen-bond network at the subunit interface. The results are used to construct a free-energy reaction coordinate that accounts for the negative cooperativity and distinctive thermodynamic signature of L-arginine binding detected by calorimetry. The symmetry of the hexamer is maintained as each ligand binds, despite the conceptual asymmetry of partially-liganded states. The results thus offer the first opportunity to describe in structural and thermodynamic terms the symmetric relaxed state predicted by the concerted allostery model of Monod, Wyman, and Changeux, revealing that this state is achieved by exploiting the dynamics of the assembly and the distributed nature of its cohesive free energy. The ArgR example reveals that symmetry can be maintained even when binding sites fill sequentially due to negative cooperativity, which was not anticipated by the Monod, Wyman, and Changeux model. The molecular mechanism identified here neither specifies nor requires a pathway for transmission of the allosteric signal through the protein, and it suggests the possibility that binding of free amino acids was an early innovation in the evolution of allostery.

Synthetic water-soluble phenolic antioxidant regulates L-arginine metabolism in macrophages: A possible role of Nrf2/ARE

Synthetic water-soluble phenolic antioxidant TS-13 exhibits pronounced anti-inflammatory properties in vivo and induces intracellular signal system Nrf2/ARE. At concentrations 150-1000 microM it inhibits nitric oxide (NO) production in mouse peritoneal macrophages. However, this compound at low concentrations (1-100 microM) paradoxically increases NO production and decreases activity of arginase. These results are indicative of an ambiguous role of NO and its metabolites in the mechanism of development of inflammatory reaction.

Spermidine Influence on the Nitric Oxide Synthesis and Arginase Activity Relationship During Experimentally Induced Seizures

Nitric oxide (NO), a potential candidate for a modulator of convulsive activity, is a mediator in several pathological events in the central nervous system. The polyamines, spermidine (Spd) and spermine, are neuromodulators influencing the metabolism of L-arginine and NO production. Here we examined the effects of Spd on NO production and arginase activity during convulsions induced by pentylenetetrazol (PTZ). Male Wistar rats were allocated into four experimental groups of 8 animals each and received the following treatments: I (control)--saline, intraperitoneally (i.p.); II (PTZ)--seizures induced by pentylenetetrazol (100mg/kg bw i.p); III (Spd)--Spd (1 mg/kg bw i.p.) 50 min before PTZ application; IV (Mid)--antiepileptic Midazolam (100 mg/kg bw) 45 min before PTZ. In brain cortex, striatum, hippocampus, cerebellum, and brainstem homogenates, nitrite + nitrate levels and arginase activity were determined. Spermidine showed proepileptic effects. shortening seizure latency and inducing a more profound increase of NO production than PTZ in all brain structures. PTZ reduced arginase activity, whereas Spd pretreatment increased enzyme activity, with the most profound effects in cerebellum and brainstem. The results point out the importance of polyamine and arginine metabolism in the brain during seizures, suggesting a regulatory role for polyamines and arginase in NO production.