Citrulline-nitric Oxide Cycle Research Articles

Citrulline depletion by ASS1 is required for proinflammatory macrophage activation and immune responses

Citrulline can be converted into argininosuccinate by argininosuccinate synthetase (ASS1) in the urea cycle and the citrulline-nitric oxide cycle. However, the regulation and biological function of citrulline metabolism remain obscure in the immune system. Unexpectedly, we found that macrophage citrulline declines rapidly after interferon gamma (IFN-γ) and/or lipopolysaccharide (LPS) stimulation, which is required for efficient proinflammatory signaling activation. Mechanistically, IFN-γ and/or LPS stimulation promotes signal transducers and activators of transcription 1 (STAT1)-mediated ASS1 transcription and Janus kinase2 (JAK2)-mediated phosphorylation of ASS1 at tyrosine 87, thereby leading to citrulline depletion. Reciprocally, increased citrulline directly binds to JAK2 and inhibits JAK2-STAT1 signaling. Blockage of ASS1-mediated citrulline depletion suppresses the host defense against bacterial infection invivo. We therefore define a central role for ASS1 in controlling inflammatory macrophage activation and antibacterial defense through depletion of cellular citrulline and, further, identify citrulline as an innate immune-signaling metabolite that engages a metabolic checkpoint for proinflammatory responses.

In vivo mechanotransduction: Effect of acute exercise on the metabolomic profiles of mouse synovial fluid

ObjectiveExercise is known to induce beneficial effects in synovial joints. However, the mechanisms underlying these are unclear. Synovial joints experience repeated mechanical loading during exercise. These mechanical stimuli are transduced into biological responses through cellular mechanotransduction. Mechanotransduction in synovial joints is typically studied in tissues. However, synovial fluid directly contacts all components of the joint, and thus may produce a whole-joint picture of the mechanotransduction response to loading. The objective of this study was to determine if metabolic phenotypes are present in the synovial fluid after acute exercise as a first step to understanding the beneficial effects of exercise on the joint. Material and methodsMice underwent a single night of voluntary wheel running or standard housing and synovial fluid was harvested for global metabolomic profiling by LC-MS. Hierarchical unsupervised clustering, partial least squares discriminant, and pathway analysis provided insight into exercise-induced mechanotransduction. ResultsAcute exercise produced a distinct metabolic phenotype in synovial fluid. Mechanosensitive metabolites included coenzyme A derivatives, prostaglandin derivatives, phospholipid species, tryptophan, methionine, vitamin D3, fatty acids, and thiocholesterol. Enrichment analysis identified several pathways previously linked to exercise including amino acid metabolism, inflammatory pathways, citrulline-nitric oxide cycle, catecholamine biosynthesis, ubiquinol biosynthesis, and phospholipid metabolism. ConclusionTo our knowledge, this is the first study to investigate metabolomic profiles of synovial fluid during in vivo mechanotransduction. These profiles indicate that exercise induced stress-response processes including both pro- and anti-inflammatory pathways. Further research will expand these results and define the relationship between the synovial fluid and the serum.

Muscle Metabolome Profiles in Woody Breast-(un)Affected Broilers: Effects of Quantum Blue Phytase-Enriched Diet.

Woody breast (WB) myopathy is significantly impacting modern broilers and is imposing a huge economic burden on the poultry industry worldwide. Yet, its etiology is not fully defined. In a previous study, we have shown that hypoxia and the activation of its upstream mediators (AKT/PI3K/mTOR) played a key role in WB myopathy, and supplementation of quantum blue (QB) can help to reduce WB severity via modulation of hypoxia-related pathways. To gain further insights, we undertook here a metabolomics approach to identify key metabolite signatures and outline their most enriched biological functions. Ultra performance liquid chromatography coupled with high resolution mass spectrometry (UPLC–HRMS) identified a total of 108 known metabolites. Of these, mean intensity differences at P < 0.05 were found in 60 metabolites with 42 higher and 18 lower in WB-affected compared to unaffected muscles. Multivariate analysis and Partial Least Squares Discriminant analysis (PLS-DA) scores plot displayed different clusters when comparing metabolites profile from affected and unaffected tissues and from moderate (MOD) and severe (SEV) WB muscles indicating that unique metabolite profiles are present for the WB-affected and unaffected muscles. To gain biologically related molecule networks, a stringent pathway analyses was conducted using IPA knowledge-base. The top 10 canonical pathways generated, using a fold-change −1.5 and 1.5 cutoff, with the 50 differentially abundant-metabolites were purine nucleotide degradation and de novo biosynthesis, sirtuin signaling pathway, citrulline-nitric oxide cycle, salvage pathways of pyrimidine DNA, IL-1 signaling, iNOS, Angiogenesis, PI3K/AKT signaling, and oxidative phosphorylation. The top altered bio-functions in term of molecular and cellular functions in WB-affected tissues included cellular development, cellular growth and proliferation, cellular death and survival, small molecular biochemistry, inflammatory response, free radical scavenging, cell signaling and cell-to-cell interaction, cell cycles, and lipid, carbohydrate, amino acid, and nucleic acid metabolisms. The top disorder functions identified were organismal injury and abnormalities, cancer, skeletal and muscular disorders, connective tissue disorders, and inflammatory diseases. Breast tissues from birds fed with high dose (2,000 FTU) of QB phytase exhibited 22 metabolites with significantly different levels compared to the control group with a clear cluster using PLS-DA analysis. Of these 22 metabolites, 9 were differentially abundant between WB-affected and unaffected muscles. Taken together, this study determined many metabolic signatures and disordered pathways, which could be regarded as new routes for discovering potential mechanisms of WB myopathy.

Oocyte specific transcription regulators, nobox and figla, are identified as key contributors to the decline in fecundity associated with ovarian aging

Advanced maternal age (AMA; ≥35 years) is associated with a decline in fecundity that is largely attributed to loss of oocyte number and quality. The aim of this study was to explore at a molecular level the relationship between aging, ovarian environment and oocyte quality. Longitudinal research study. Young outbred CD1 female mice (3-4 months old; Young) and naturally aged outbred CD1 female mice (10-12 months old; Aged) were super-ovulated and oocytes collected (n = 10 group) for quantitative immunofluorescence of endoplasmic reticulum (ER) stress indicators, pIRE1 and ATF6. Total RNA was isolated from unstimulated ovaries (n = 6 per group), sequence libraries were prepared using the TruSEQ Total RNA library kit (Illumina) and sequenced on the Illumina NovaSEQ 6000. Differentially expressed genes (DEGs) were generated using edgeR, with an FDR cutoff of 5% (q value <0.01) and Student’s t-test significance at p<0.001, followed by Ingenuity Pathway Analysis (Qiagen). Oocyte numbers significantly declined with natural aging (mean: Aged = 4.6, Young = 12.9; p<0.0001). Total RNA sequencing revealed 281 significant DEGs in Aged versus Young ovaries (120 increased and 161 decreased; p<0.0001). Unsupervised hierarchical clustering of the 281 DEGs cleanly separated the ovaries according to female age. Enriched pathway analysis revealed signaling pathways including Citrulline-Nitric Oxide Cycle, VEGF Family Ligand Receptor Interactions, and HIF1α signaling. Nitric oxide is a common signaling molecule in these pathways, and has been shown to maintain diplotene arrest in pre-ovulatory oocytes. Aged ovaries displayed a significant decrease in nitric oxide gene expression (p<0.0001) that could lead to premature meiotic resumption. Interestingly, a significant increase in the proportion of immature oocytes was also observed with natural aging (mean: Aged = 79.1%, Young = 24.0%; p<0.0001). Oocyte specific upstream transcription factors, NOBOX and FIGLA, were identified as significantly inhibited in Aged ovaries (p<0.0001). Vital oocyte genes targeted by these regulators include Gdf9 (folliculogenesis), H1foo (germinal vesicle maturation), Rfpl4 (oogenesis) and Zp3 (zona pellucida glycoprotein), all significantly decreased in Aged ovaries (P<0.0001). Additionally, FIGLA plays a role promoting ubiquitin-mediated proteolysis via genes Fbxw21 and Nlrp4f, both significantly decreased in Aged ovaries (p<0.0001). Inhibition of protein degradation results in cellular stress identified by increased intensity of staining and nuclear localization of ER stress indicators, ATF6 (p<0.01) and pIRE1 (p<0.05), in Aged oocytes. This study revealed at a molecular level the widespread, damaging impacts of natural aging on ovarian function, including significant transcriptomic and protein expression changes that directly contribute to the decline in oocyte quality and overall fecundity. Ongoing studies are focused on manipulating identified genomic targets with the potential to slow down ovarian aging.

Effect of prematurity on genome wide methylation in the placenta

BackgroundPreterm birth is a significant clinical problem and an enormous burden on society, affecting one in eight pregnant women and their newborns. Despite decades of research, the molecular mechanism underlying its pathogenesis remains unclear. Many studies have shown that preterm birth is associated with health risks across the later life course. The “fetal origins” hypothesis postulates that adverse intrauterine exposures are associated with later disease susceptibility. Our recent studies have focused on the placental epigenome at term. We extended these studies to genome-wide placental DNA methylation across a wide range of gestational ages. We applied methylation dependent immunoprecipitation/DNA sequencing (MeDIP-seq) to 9 placentas with gestational age from 25 weeks to term to identify differentially methylated regions (DMRs).ResultsEnrichment analysis revealed 427 DMRs with nominally significant differences in methylation between preterm and term placentas (p < 0.01) and 21 statistically significant DMRs after multiple comparison correction (FDR p < 0.05), of which 62% were hypo-methylated in preterm placentas vs term placentas. The majority of DMRs were in distal intergenic regions and introns. Significantly enriched pathways identified by Ingenuity Pathway Analysis (IPA) included Citrulline-Nitric Oxide Cycle and Fcy Receptor Mediated Phagocytosis in macrophages. The DMR gene set overlapped placental gene expression data, genes and pathways associated evolutionarily with preterm birth.ConclusionThese studies form the basis for future studies on the epigenetics of preterm birth, “fetal programming” and the impact of environment exposures on this important clinical challenge.

PRMT7 Interacts with ASS1 and Citrullinemia Mutations Disrupt the Interaction

Protein arginine methyltransferase 7 (PRMT7) catalyzes the introduction of monomethylation marks at the arginine residues of substrate proteins. PRMT7 plays important roles in the regulation of gene expression, splicing, DNA damage, paternal imprinting, cancer and metastasis. However, little is known about the interaction partners of PRMT7. To address this, we performed yeast two-hybrid screening of PRMT7 and identified argininosuccinate synthetase (ASS1) as a potential interaction partner of PRMT7. We confirmed that PRMT7 directly interacts with ASS1 using pull-down studies. ASS1 catalyzes the rate-limiting step of arginine synthesis in urea cycle and citrulline-nitric oxide cycle. We mapped the interface of PRMT7–ASS1 complex through computational approaches and validated the predicted interface in vivo by site-directed mutagenesis. Evolutionary analysis revealed that the ASS1 residues important for PRMT7–ASS1 interaction have co-evolved with PRMT7. We showed that ASS1 mutations linked to type I citrullinemia disrupt the ASS1–PRMT7 interaction, which might explain the molecular pathogenesis of the disease.

Immunohistochemical Analysis of Citrulline-Nitric Oxide Cycle Enzymes and Glutamine Synthetase in Different Regions of Brain in Epilepsy Rat Model

The aim of this study was to determine the immunoreactivity of neuronal and inducible nitric oxide synthetase, argininosuccinate synthetase, argininosuccinate lyase, glutamine synthetase in different regions of brain in rats of kainic acid mediated epilepsy. Male Sprague-Dawley rats were used in this study. The acute group animals were sacrificed after 2 hours and the chronic group animals were sacrificed after 5 days of a single subcutaneous injection of kainic acid (15 mg/kg body weight). The cerebral cortex, cerebellum and brain stem slices were fixed and immunohistostained for the above enzymes. Images were captured and analyzed. In acute group, argininosuccinate synthetase and inducible nitric oxide synthetase were increased in cerebral cortex and cerebellum, neuronal nitric oxide synthetase increased in cerebral cortex and brain stem, and there was no change in argininosuccinate lyase immunoreactivity compared to control group. In chronic group, glutamine synthetase was decreased and all other enzymes immunoreactivity was increased in all the brain regions tested. This study demonstrated the up-regulation of citrul-line-nitric oxide cycle enzymes and may contribute to enhancing recycling of citrulline to arginine to support the increased production of nitric oxide in epilepsy. The decreased glutamine synthetase may increase glutamate in chronic epilepsy and may lead to neurodegeneration.

Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function

Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline–NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regulates AS and eNOS transcription to support NO production. Moreover, we show that insulin enhances NO production in response to physiological cues such as bradykinin. To translate these results to an in vivo model, we show that AS transcription is diminished in coronary endothelial cells isolated from rats with streptozotocin (STZ)-induced diabetes. Importantly, we demonstrate restoration of AS and eNOS transcription by insulin treatment in STZ-diabetic rats, and show that this restoration was accompanied by improved endothelial function as measured by endothelium-dependent vasorelaxation. Overall, this report demonstrates, both in cell culture and whole animal studies, that insulin maintains vascular function, in part, through the maintenance of AS transcription, thus ensuring an adequate supply of arginine to maintain vascular endothelial response to physiological cues.

English

The aim of this study was to determine the mRNA expression of nitric oxide synthetase (NOS, argininosuccinate synthetase (AS), argininosuccinate lyase (AL) and glutamine synthetase (GS) in different regions of brain in rats subjected to kainic acid (KA) mediated epilepsy. The short term (acute) group animals were sacrificed after 2 h and the long term (chronic) group animals were sacrificed after 5 days of single injection of KA. After decapitation of rats, cerebral cortex (CC), cerebellum (CB) and brain stem (BS) were separated and in their homogenates, the relative amount of nNOS, iNOS, AS, AL and GS mRNA was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Results showed an increased expression of iNOS in all brain regions tested in chronic group as compared to either control or acute group, and it indicate a favorable condition of nitric oxide production. AL expression was significantly increased only in CB in acute group whereas in chronic group it is increased in CC and CB and decreased in BS as compared to control. The aforementioned increased expression of AL may contribute effective recycling of citrulline to arginine. No change in expression of nNOS and AS in both acute and chronic groups of epilepsy. GS expression was significantly decreased only in chronic group of epilepsy in all brain regions tested when compared with control group. The decreased GS may be contributing prolonged availability of glutamate in chronic epilepsy. &nbsp; Key words:&nbsp;Citrulline-nitric oxide cycle enzymes, glutamine synthetase, reverse transcriptase-polymerase chain reaction (RT-PCR), epilepsy.

Enhancement of the citrulline–nitric oxide cycle in astroglioma cells by the proline-rich peptide-10c from Bothrops jararaca venom

The biological activity of the proline-rich decapeptide Bj-PRO-10c, a processing product of the C-type natriuretic peptide precursor protein, expressed in the brain and the venom gland of the pit viper Bothrops jararaca, was originally attributed to the inhibition of the somatic angiotensin-converting enzyme activity with subsequent anti-hypertensive effect. However, recent results suggest broader biological activity may also be involved in the cardiovascular effects of this peptide. Here we show that Bj-PRO-10c enhances and sustains the generation of nitric oxide (NO) by regulating argininosuccinate synthase activity and thereby velocity of the citrulline–NO cycle. Bj-PRO-10c-mediated effects not restricted to the cardiovascular system, since NO production was also induced in cells of astroglial origin. Bj-PRO-10c was internalized by C6 astroglioma cells where it induces NO production and upregulation of the citrulline–NO cycle cells in a dose-dependent fashion. In view of that, astroglial cells function as l-arginine pool for NO production in neighboring neurons, we suggest a regulatory function for Bj-PRO-10c on the metabolism of this gaseous neurotransmitter in the CNS. Moreover, proliferation of astroglial cells was reduced in the presence of Bj-PRO-10c; however, cell death was not induced. Since NO donors have been studied for the treatment of solid cancers, Bj-PRO-10c may serve as structural model for developing drugs to improve the effects of cancer therapy based on the peptide's ability to augment NO production.

Nitric oxide (NO), citrulline–NO cycle enzymes, glutamine synthetase, and oxidative status in kainic acid–mediated excitotoxicity in rat brain

Neuronal excitation, involving the excitatory glutamate receptors, is recognized as an important underlying mechanism in neurodegenerative disorders. To understand their role in excitotoxicity, the nitric oxide synthase (NOS), argininosuccinate synthetase (AS), argininosuccinate lyase (AL), glutamine synthetase (GS), and arginase activities, along with the concentration of nitrate/nitrite, thiobarbituric acid–reactive substances (TBARS), and total antioxidant status (TAS), were estimated in the cerebral cortex, cerebellum, and brain stem of rats subjected to kainic acid–mediated excitotoxicity. The results of this study clearly demonstrated the increased production of NO by increased activity of NOS. The increased activities of AS and AL suggest the increased and effective recycling of citrulline to arginine in excitotoxicity, making NO production more effective and contributing to its toxic effects. The decreased activity of GS may favor the prolonged availability of glutamic acid, causing excitotoxicity, leading to neuronal damage. The increased formation of TBARS and decreased TAS indicate the presence of oxidative stress in excitotoxicity.

Characterization of AS Regulatory Protein, a Novel Protein Implicated in Endothelial Stress Response

The citrulline‐nitric oxide cycle is a tightly regulated system for the production of nitric oxide (NO) in endothelial cells. The cycle consists of the proteins endothelial nitric oxide synthase (eNOS), argininosuccinate synthase (AS), and argininosuccinate lyase (AL). AS and AL serve to recycle citrulline, a product of the production of NO, to arginine, the principle substrate for eNOS. AS regulatory protein (ARP) is a small protein encoded by an upstream open reading frame in minor expressed AS mRNAs. Overexpression of ARP suppressed AS expression and NO production in endothelial cells while selective knockdown of the longer AS transcripts that produce ARP resulted in increased AS protein expression and NO production. To investigate the subcellular localization of ARP, a custom polyclonal antibody was generated. Fluorescence microscopy studies demonstrated that ARP is expressed throughout the cytoplasm but that expression seems predominant in the perinuclear endoplasmic reticulum and golgi, co‐localizing with both eNOS and AS. Co‐immunostaining for ARP and TIA‐1, a marker for stress granule formation, demonstrated that ARP moves to stress granules when stress is induced artificially by sodium arsenite in endothelial cells. This data demonstrates that ARP may play a role in the sequestration of mRNAs under conditions of stress. (Supported by the James &amp; Esther King Biomedical Research Program Grant).

Troglitazone up-regulates vascular endothelial argininosuccinate synthase

Vascular endothelial nitric oxide (NO) production via the citrulline–NO cycle not only involves the regulation of endothelial nitric oxide synthase (eNOS), but also regulation of caveolar-localized endothelial argininosuccinate synthase (AS), which catalyzes the rate-limiting step of the cycle. In the present study, we demonstrated that exposure of endothelial cells to troglitazone coordinately induced AS expression and NO production. Western blot analysis demonstrated an increase in AS protein expression. This increased expression was due to transcriptional upregulation of AS mRNA, as determined by quantitative real time RT-PCR and inhibition by 1-d-ribofuranosylbenzimidazole (DRB), a transcriptional inhibitor. Reporter gene assays and EMSA analyses identified a distal PPARγ response element (PPRE) (−2471 to −2458) that mediated the troglitazone increase in AS expression. Overall, this study defines a novel molecular mechanism through which a thiazolidinedione (TZD) like troglitazone supports endothelial function via the transcriptional up-regulation of AS expression.

Hypoxia Impairs the Citrulline‐Nitric Oxide Cycle in Aortic Endothelial Cells

Hypoxia, a condition caused by a significant and prolonged decrease in oxygen, results in endothelial dysfunction, in part, due to the impairment of nitric oxide (NO) production. Endothelial NO is an “endogenous vasodilator” signaling vascular smooth muscle relaxation which lowers resistance to blood flow and consequently decreases blood pressure. Previous studies have shown that under hypoxic conditions, endothelial nitric oxide synthase (eNOS) expression is reduced in bovine aortic endothelial cells (BAEC). Our laboratory has shown that endothelial argininosuccinate synthase (AS) is essential for NO production, providing arginine, the substrate for eNOS. Thus, we investigated whether hypoxia induced a coordinate suppression of both eNOS and AS, resulting in impaired NO production. Initially, cobalt chloride or desferrioxamine treatment of BAEC was used to simulate hypoxic conditions. In each case, western blot analysis demonstrated a coordinate and dose-dependent decrease in AS and eNOS protein expression. Subsequently, the direct effect of hypoxia was investigated by culturing BAEC under 2% O2 conditions. In this case, quantitative real-time PCR demonstrated a significant reduction of AS and eNOS mRNAs, and western blot analysis showed a corresponding decrease in protein. In conclusion, hypoxia induces the coordinate suppression of AS and eNOS, thus compromising the citrulline-nitric oxide cycle in endothelial cells by reducing the availability of substrate, as well as eNOS activity, which results in impaired endothelial NO production.

Effects of acute ammonia toxicity on nitric oxide (NO), citrulline–NO cycle enzymes, arginase and related metabolites in different regions of rat brain

Nitric oxide (NO) is involved in many pathophysiological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS) enzymes. Citrulline formed as a by-product of the NOS reaction, can be recycled to arginine by successive actions of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) via the citrulline–NO cycle. Hyperammonemia is known to cause poorly understood perturbations of the citrulline–NO cycle. To understand the role of citrulline–NO cycle in hyperammonemia, NOS, ASS, ASL and arginase activities, as well as nitrate/nitrite (NOx), arginine, ornithine, citrulline, glutamine, gluamate and GABA were estimated in cerebral cortex (CC), cerebellum (CB) and brain stem (BS) of rats subjected to acute ammonia toxicity. NOx concentration and NOS activity were found to increase in all the regions of brain in acute ammonia toxicity. The activities of ASS and ASL showed an increasing trend whereas the arginase was not changed. The results of this study clearly demonstrated the increased formation of NO, suggesting the involvement of NO in the pathophysiology of acute ammonia toxicity. The increased activities of ASS and ASL suggest the increased and effective recycling of citrulline to arginine in acute ammonia toxicity, making NO production more effective and contributing to its toxic effects.

Induction of citrulline–nitric oxide (NO) cycle enzymes and NO production in immunostimulated rat RPE-J cells

Nitric oxide (NO) has been implicated in many physiological and pathological conditions in the eyes. The induction of inducible NO synthase (iNOS) and NO production have been noted in immunostimulated retinal pigment epithelial (RPE) cells. Cellular NO production depends on the availability of arginine, a substrate for NOS. Arginine can be regenerated from citrulline, another product of the NOS reaction, by argininosuccinate synthetase and argininosuccinate lyase, forming the citrulline–NO cycle. When rat RPE-J cells were treated with interferon-γ (IFNγ), tumor necrosis factor-α (TNFα) and lipopolysaccharide (LPS), and expression of the citrulline–NO cycle enzymes and related enzymes was analyzed, iNOS and argininosuccinate synthetase were highly induced at both mRNA and protein levels. On the other hand, argininosuccinate lyase was not induced. Among other related enzymes and transporters, mRNA for cationic amino acid transporter (CAT)-1 was weakly induced, whereas those for CAT-2, arginase I and II, ornithine aminotransferase and ornithine decarboxylase remained little changed. NO was produced by cells after stimulation with TNFα, IFNγ and LPS. The induction of iNOS mRNA and the production of NO by these immunostimulated cells was further enhanced by cAMP. NO was produced from citrulline as well as from arginine. Our findings indicate that in activated RPE-J cells citrulline–arginine recycling is important for NO production.

Coinduction of Endothelial Nitric Oxide Synthase and Arginine Recycling Enzymes in Aorta of Diabetic Rats

Decreased availability of arginine and impaired production of NO (nitric oxide) have been implicated in the development of endothelial dysfunction. Citrulline formed by the NOS reaction is recycled to arginine by the citrulline–NO cycle, which is composed of NOS, argininosuccinate synthetase (AS), and argininosuccinate lyase. Therefore, we investigated the alterations of these enzymes in the aorta of streptozotocin (STZ)-induced diabetic rats. eNOS and AS mRNAs were increased by three- to fourfold 1–2 weeks after STZ treatment and decreased at 4 weeks. AL mRNA was weakly induced. Induction of eNOS and AS proteins was also observed. Cationic amino acid transporter (CAT)-1 mRNA remained little changed, and CAT-2 mRNA was not detected. The plasma nitrogen oxide levels were increased 1–2 weeks after STZ treatment and decreased at 4 weeks. Transforming growth factor-β1 (TGF-β1) mRNA in the aorta was also induced. TGF-β1 induced eNOS and AS mRNAs in human umbilical vein endothelial cells but inhibited the proliferation of HUVEC. These results indicate that eNOS and AS are coinduced in the aorta in early stages of STZ-induced diabetic rats and that the induction is mediated by TGF-β1. The results also suggest that TGF-β1 works antiatherogenically at early stages of diabetes by increasing NO production, whereas prolonged elevation of TGF-β1 functions atherogenically by inhibiting endothelial cell growth.

Coinduction of inducible nitric oxide synthase and arginine recycling enzymes in cytokine-stimulated PC12 cells and high output production of nitric oxide

Nitric oxide (NO) is involved in many physiological and pathological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS), and the citrulline generated as a by-product can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) via the citrulline–NO cycle. When neuronal PC12 cells differentiated with nerve growth factor were treated with interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα), iNOS and AS mRNAs and proteins were markedly induced, with AL mRNA and protein being weakly induced. Cationic amino acid transporter-1 and -2 were not induced. IFNγ or TNFα alone was ineffective. A large amount of NO (190 μM NO 2 − plus NO 3 − in culture medium in 24 h) was produced from arginine by cytokine-stimulated cells, and arginine could be replaced by citrulline. iNOS induction and NO production were attenuated by dexamethasone and dibutyryl cAMP and even more strongly so when combined. Therefore, a large amount of NO is produced in cytokine-stimulated PC12 cells following to induction of iNOS and citrulline-arginine recycling is important for NO production.

Expression of citrulline–nitric oxide cycle in lipopolysaccharide and cytokine-stimulated rat astroglioma C6 cells

Nitric oxide (NO) is involved in many physiological and pathological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS), with citrulline generated as a by-product of the reaction. Thus, citrulline can by recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) via the citrulline–NO cycle. Rat astroglioma C6 cells were treated with bacterial lipopolysaccharide (LPS), interferon-γ (IFNγ) and tumor necrosis factor-α, and the expression of the enzymes of the citrulline–NO cycle was investigated by RNA blot and immunoblot analyses. NO production from arginine and citrulline was also assessed. iNOS mRNA and protein were induced 6–12 h after stimulation with LPS and cytokines and decreased at 24 h. AS mRNA increased up to 12 h and decreased at 24 h. AS protein increased gradually up to 48 h. On the other hand, AL mRNA remained unchanged by stimulation. NO production from arginine was enhanced by the treatment with LPS and cytokines. NO production was also observed when arginine was replaced by citrulline. These results indicate that NO production is enhanced in LPS- and cytokine-stimulated C6 cells due to induction of iNOS and that the citrulline–arginine recycling is important for NO production.

L-arginine uptake, the citrulline–NO cycle and arginase II in the rat brain: an in situ hybridization study

Nitric oxide (NO) is synthesized from a unique precursor, arginine, by nitric oxide synthase (NOS). In brain cells, arginine is supplied by protein breakdown or extracted from the blood through cationic amino acid transporters (CATs). Arginine can also be recycled from the citrulline produced by NOS activity, through argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) activities, and metabolized by arginase. NOS, AS and AL constitute the so-called citrulline–NO cycle. In order to better understand arginine transport, recycling and degradation, we studied the regional distribution of cells expressing CAT1, CAT3, AS, AL, neuronal NOS (nNOS) and arginase II (AII) in the adult rat brain by non-radioisotopic in situ hybridization (ISH). CAT1, AL and AII presented an ubiquitous neuronal and glial expression, whereas CAT3 and AS were confined to neurons. nNOS was restricted to scattered neurons and a few brain nuclei and layers. We demonstrate by this study that cells expressing nNOS all appear to express the entire citrulline–NO cycle, whereas numerous cells expressing AL do not express AS. The differential expression of these genes within the same anatomical structure could indicate that intercellular exchanges of citrulline–NO cycle metabolites are relevant. Thus vicinal interactions should be taken into account to study their regulatory mechanisms.