Macrophage fumarate hydratase restrains mtRNA-mediated interferon production.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

Metabolic reprograming of LPS-stimulated human lung macrophages involves tryptophan metabolism and the aspartate-arginosuccinate shunt.

Lung macrophages (LM) are in the first line of defense against inhaled pathogens and can undergo phenotypic polarization to the proinflammatory M1 after stimulation with Toll-like receptor agonists. The objective of the present work was to characterize the metabolic alterations occurring during the experimental M1 LM polarization. Human LM were obtained from resected lungs and cultured for 24 hrs in medium alone or with 10 ng.mL-1 lipopolysaccharide. Cells and culture supernatants were subjected to extraction for metabolomic analysis with high-resolution LC-MS (HILIC and reverse phase -RP- chromatography in both negative and positive ionization modes) and GC-MS. The data were analyzed with R and the Worklow4Metabolomics and MetaboAnalyst online infrastructures. A total of 8,741 and 4,356 features were detected in the intracellular and extracellular content, respectively, after the filtering steps. Pathway analysis showed involvement of arachidonic acid metabolism, tryptophan metabolism and Krebs cycle in the response of LM to LPS, which was confirmed by the specific quantitation of selected compounds. This refined analysis highlighted a regulation of the kynurenin pathway as well as the serotonin biosynthesis pathway, and an involvement of aspartate-arginosuccinate shunt in the malate production. Macrophages M1 polarization is accompanied by changes in the cell metabolome, with the differential expression of metabolites involved in the promotion and regulation of inflammation and antimicrobial activity. The analysis of this macrophage immunometabolome may be of interest for the understanding of the pathophysiology of lung inflammatory disesases.

Global metabolomic profiling of uterine leiomyomas.

BACKGROUND: Uterine leiomyomas can be classified into molecularly distinct subtypes according to their genetic triggers: MED12 mutations, HMGA2 upregulation, or inactivation of FH. The aim of this study was to identify metabolites and metabolic pathways that are dysregulated in different subtypes of leiomyomas. METHODS: We performed global metabolomic profiling of 25 uterine leiomyomas and 17 corresponding myometrium specimens using liquid chromatography-tandem mass spectroscopy. RESULTS: A total of 641 metabolites were detected. All leiomyomas displayed reduced homocarnosine and haeme metabolite levels. We identified a clearly distinct metabolomic profile for leiomyomas of the FH subtype, characterised by metabolic alterations in the tricarboxylic acid cycle and pentose phosphate pathways, and increased levels of multiple lipids and amino acids. Several metabolites were uniquely elevated in leiomyomas of the FH subtype, including N6-succinyladenosine and argininosuccinate, serving as potential biomarkers for FH deficiency. In contrast, leiomyomas of the MED12 subtype displayed reduced levels of vitamin A, multiple membrane lipids and amino acids, and dysregulation of vitamin C metabolism, a finding which was also compatible with gene expression data. CONCLUSIONS: The study reveals the metabolomic heterogeneity of leiomyomas and provides the requisite framework for strategies designed to target metabolic alterations promoting the growth of these prevalent tumours.

Biochemical characterization of argininosuccinate lyase from M. tuberculosis: significance of a c-terminal cysteine in catalysis and thermal stability.

Arginine biosynthesis pathway is crucial to the survival and pathogenesis of Mycobacterium tuberculosis (Mtb). Arginine is a critical amino acid that contributes to the inflection of cellular immune responses during pathogenesis. Argininosuccinate lyase from Mtb (MtArgH), the last enzyme in the pathway, catalyzes the production of arginine from argininosuccinic acid. MtArgH is an essential enzyme for the growth and survival of M. tuberculosis. We biochemically characterized MtArgH and deciphered the role of a previously unexplored cysteine (Cys441 ) residue at the C-terminal region of the protein. Chemical modification of Cys441 completely abrogated the enzymatic activity suggesting its involvement in the catalytic mechanism. Replacement of Cys441 to alanine showed a striking decrease in the enzymatic activity, while retaining the overall secondary to quaternary structure of the protein, hence corroborating the involvement of Cys441 in the process of catalysis. Interestingly, replacement of Cys441 to serine, showed significant increase in activity, as compared to the wild-type MtArgH. Inactivity of C441 A and elevated activity of its conservative mutant (C441 S) confirmed the participation of Cys441 in the MtArgH activity. We also, observed that C441 S mutant has higher thermal stability and maintains significant activity at high temperatures. This is in concordance with our observation that Cys441 in Mtb is replaced by a serine in the ArgH from thermophilic microorganisms. Furthermore, we also propose a potential feedback mechanism, wherein the Cys441 is covalently modified to S-(2-succinyl) cysteine (succination) by one of the products, fumarate, thereby inactivating MtArgH. These insights into the mechanism of MtArgH activity unravel novel regulations of arginine biosynthetic pathway in Mtb. © 2017 IUBMB Life, 69(11):896-907, 2017.

Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization.

Macrophage polarization involves a coordinated metabolic and transcriptional rewiring that is only partially understood. By using an integrated high-throughput transcriptional-metabolic profiling and analysis pipeline, we characterized systemic changes during murine macrophage M1 and M2 polarization. M2 polarization was found to activate glutamine catabolism and UDP-GlcNAc-associated modules. Correspondingly, glutamine deprivation or inhibition of N-glycosylation decreased M2 polarization and production of chemokine CCL22. In M1 macrophages, we identified a metabolic break at Idh, the enzyme that converts isocitrate to alpha-ketoglutarate, providing mechanistic explanation for TCA cycle fragmentation. (13)C-tracer studies suggested the presence of an active variant of the aspartate-arginosuccinate shunt that compensated for this break. Consistently, inhibition of aspartate-aminotransferase, a key enzyme of the shunt, inhibited nitric oxide and interleukin-6 production in M1 macrophages, while promoting mitochondrial respiration. This systems approach provides a highly integrated picture of the physiological modules supporting macrophage polarization, identifying potential pharmacologic control points for both macrophage phenotypes.

A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia.

The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

Argininosuccinate lyase deficiency.

The urea cycle consists of six consecutive enzymatic reactions that convert waste nitrogen into urea. Deficiencies of any of these enzymes of the cycle result in urea cycle disorders (UCDs), a group of inborn errors of hepatic metabolism that often result in life-threatening hyperammonemia. Argininosuccinate lyase (ASL) catalyzes the fourth reaction in this cycle, resulting in the breakdown of argininosuccinic acid to arginine and fumarate. ASL deficiency (ASLD) is the second most common UCD, with a prevalence of ~1 in 70,000 live births. ASLD can manifest as either a severe neonatal-onset form with hyperammonemia within the first few days after birth or as a late-onset form with episodic hyperammonemia and/or long-term complications that include liver dysfunction, neurocognitive deficits, and hypertension. These long-term complications can occur in the absence of hyperammonemic episodes, implying that ASL has functions outside of its role in ureagenesis and the tissue-specific lack of ASL may be responsible for these manifestations. The biochemical diagnosis of ASLD is typically established with elevation of plasma citrulline together with elevated argininosuccinic acid in the plasma or urine. Molecular genetic testing of ASL and assay of ASL enzyme activity are helpful when the biochemical findings are equivocal. However, there is no correlation between the genotype or enzyme activity and clinical outcome. Treatment of acute metabolic decompensations with hyperammonemia involves discontinuing oral protein intake, supplementing oral intake with intravenous lipids and/or glucose, and use of intravenous arginine and nitrogen-scavenging therapy. Dietary restriction of protein and dietary supplementation with arginine are the mainstays in long-term management. Orthotopic liver transplantation (OLT) is best considered only in patients with recurrent hyperammonemia or metabolic decompensations resistant to conventional medical therapy.

Argininosuccinate lyase deficiency-argininosuccinic aciduria and beyond.

The urea cycle consists of six consecutive enzymatic reactions that convert waste nitrogen into urea. Deficiencies of any of these enzymes of the cycle result in urea cycle disorders (UCD), a group of inborn errors of hepatic metabolism that often result in life threatening hyperammonemia. Argininosuccinate lyase (ASL) is a cytosolic enzyme which catalyzes the fourth reaction in the cycle and the first degradative step, that is, the breakdown of argininosuccinic acid to arginine and fumarate. Deficiency of ASL results in an accumulation of argininosuccinic acid in tissues, and excretion of argininosuccinic acid in urine leading to the condition argininosuccinic aciduria (ASA). ASA is an autosomal recessive disorder and is the second most common UCD. In addition to the accumulation of argininosuccinic acid, ASL deficiency results in decreased synthesis of arginine, a feature common to all UCDs except argininemia. Arginine is not only the precursor for the synthesis of urea and ornithine as part of the urea cycle but it is also the substrate for the synthesis of nitric oxide, polyamines, proline, glutamate, creatine, and agmatine. Hence, while ASL is the only enzyme in the body able to generate arginine, at least four enzymes use arginine as substrate: arginine decarboxylase, arginase, nitric oxide synthetase (NOS) and arginine/glycine aminotransferase. In the liver, the main function of ASL is ureagenesis, and hence, there is no net synthesis of arginine. In contrast, in most other tissues, its role is to generate arginine that is designated for the specific cell's needs. While patients with ASA share the acute clinical phenotype of hyperammonemia, encephalopathy, and respiratory alkalosis common to other UCD, they also present with unique chronic complications most probably caused by a combination of tissue specific deficiency of arginine and/or elevation of argininosuccinic acid. This review article summarizes the clinical characterization, biochemical, enzymatic, and molecular features of this disorder. Current treatment, prenatal diagnosis, diagnosis through the newborn screening as well as hypothesis driven future treatment modalities are discussed.

Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts.

There are many reports of an arginine-dependent nitric oxide synthase activity in plants; however, the gene(s) or protein(s) responsible for this activity have yet to be convincingly identified. To measure nitric oxide synthase activity, many studies have relied on a citrulline-based assay that measures the formation of L-citrulline from L-arginine using ion exchange chromatography. In this article, we report that when such assays are used with protein extracts from Arabidopsis, an arginine-dependent activity was observed, but it produced a product other than citrulline. TLC analysis identified the product as argininosuccinate. The reaction was stimulated by fumarate (> 500 microM), implicating the urea cycle enzyme argininosuccinate lyase (EC 4.3.2.1), which reversibly converts arginine and fumarate to argininosuccinate. These results indicate that caution is needed when using standard citrulline-based assays to measure nitric oxide synthase activity in plant extracts, and highlight the importance of verifying the identity of the product as citrulline.

Ubiquitous presence of argininosuccinate at millimolar levels in the central nervous system of Aplysia californica.

Endogenous nitric oxide (NO) is generated by nitric oxide synthases (NOSs), which convert arginine (Arg) and oxygen to citrulline (Cit) and NO. Cit can be enzymatically transformed back to Arg by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) via a pathway involving argininosuccinate (ArgSuc). Arg, Cit, and ArgSuc levels have been measured in single neurons, neuronal clusters, and neuropil from the nervous system of the common neurobiological model Aplysia californica. Using capillary electrophoresis with laser-induced fluorescence detection, ArgSuc was found to be present in the nervous system in millimolar concentrations at levels significantly exceeding Cit levels (p<0.01). ArgSuc levels are proportional to Arg concentrations in single neurons, whereas they have no clear correlation to the Cit or Arg/Cit ratio. NOS-expressing neurons often exhibit fixative-resistant nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining. Incubation of ganglia with Arg results in an increase in Cit and ArgSuc levels in the NADPH-d-positive neuropil with no effect on ArgSuc levels in NADPH-d-negative neurons, suggesting NOS activity in the neuropil. Similar incubation with Cit leads to decreased ArgSuc levels in NADPH-d-negative neurons. These results can be explained by localization of NOS and ASS in different neurons; therefore, the complete Arg-Cit-NO cycle may not be present in the same neuron. The surprisingly high intracellular ArgSuc concentration suggests alternative sources of ArgSuc and that at least a portion may be formed by the reverse reaction of ASL (catalyzing the conversion of Arg to ArgSuc), which can be inhibited by Cit.

Integrity and stability of the citrulline-arginine pathway in normal and tumour cell lines.

Arginine catabolizing enzymes have been used on cancers for over 60 years. In the last 5 years the ability of arginine catabolizing enzymes, not only to inhibit proliferation, but to kill tumour cells has been reinvestigated. Selectivity of action lies in the inability of many tumours to circumvent arginine deprivation by recycling precursors through the urea cycle. While this offers an immediate window of opportunity to treat, e.g. melanomas and hepatocellular carcinomas (HCC) that have poor citrulline converting ability, it is possible that the deprivation can be applied to many other types of cancer. The problem of deficiency of the urea cycle enzymes in a wider range of normal and malignant cell lines has been addressed, and shown to be variable throughout several different tumour types. We also need to know how fickle recycling enzyme activity can be in both normal and tumour cells, and found to be remarkable stable. Increasing interest is shown in the amino acid (arginine) deprivation protocol because it has already moved into the clinic. Initial findings on a named-patient basis have been encouraging, and the development of a new rational approach to the systemic treatment of melanomas, HCCs and leukemias seems imminent. This is the more attractive because arginine deprivation protocols can also 'stage' tumour cells for combination therapy in cases where they might not be killed outright by deprivation alone.

Direct single cell determination of nitric oxide synthase related metabolites in identified nitrergic neurons.

The biochemical characterization of individual nitrergic (NO releasing) neurons is a non-trivial task both in vertebrate and invertebrate preparations. In spite of numerous efforts, there are limited data related to intracellular concentrations of essential metabolites involved in NO synthesis and degradation. This situation creates controversies in both identification of nitrergic neurons and the selection of reliable reporters of NOS activity in heterogeneous cell populations. We take advantage of identified neurons from the pulmonate mollusc Lymnaea stagnalis to perform direct single cell microanalysis of intracellular concentrations of the major nitric oxide synthase (NOS) related metabolites such as arginine, citrulline, argininosuccinate, NO(2)(-),and NO(3)(-). Capillary electrophoresis protocols have been developed to quantitate levels of these metabolites in single identified neurons from the buccal, cerebral, and pedal ganglia using laser-induced fluorescence and conductivity detection. The limits of detection (LODs) for arginine (Arg) and citrulline (Cit) are 84 amol (11nM) and 110 amol (15 nM), respectively, and LODs for NO(2)(-)and NO(3)(-) are <200 amol (<10nM) each. We report that intracellular concentrations of NOS related metabolites are in the millimolar range and less than 1% of a single cell is required for microchemical analysis. From four cell types tested, only the esophageal motoneuron B2 contains active NOS, and they also contain surprisingly high nitrite levels (up to 5mM) compared to other neurons tested (peptidergic B4, dopaminergic RPeD1, and serotonergic CGC). These B2 neurons also exhibit an Arg/Cit ratio susceptible to the selective NOS inhibitor l-iminoethyl-N-ornithine whereas others neurons do not even though they all may contain NOS transcripts. On the contrary, we found that absolute concentrations of other NOS related metabolites including nitrates are not reliable markers of NOS activity and demonstrate the need for multiple assays for NOS activity.

A duck delta1 crystallin double loop mutant provides insight into residues important for argininosuccinate lyase activity.

Delta-crystallin is directly related to argininosuccinate lyase (ASL), and catalyzes the reversible hydrolysis of argininosuccinate to arginine and fumarate. Two delta-crystallin isoforms exist in duck lenses, delta1 and delta2, which are 94% identical in amino acid sequence. Although the sequences of duck delta2-crystallin (ddeltac2) and duck delta1-crystallin (ddeltac1) are 69 and 71% identical to that of human ASL, respectively, only ddeltac2 has maintained ASL activity. Domain exchange experiments and comparisons of various delta-crystallin structures have suggested that the amino acid substitutions in the 20's (residues 22-31) and 70's (residues 74-89) loops of ddeltac1 are responsible for the loss of enzyme activity in this isoform. To test this hypothesis, a double loop mutant (DLM) of ddeltac1 was constructed in which all the residues that differ between the two isoforms in the 20's and 70's loops were mutated to those of ddeltac2. Contrary to expectations, kinetic analysis of the DLM found that it was enzymatically inactive. Furthermore, binding of argininosuccinate by the DLM, as well as the ddeltac1, could not be detected by isothermal titration calorimetry (ITC). To examine the conformation of the 20's and 70's loops in the DLM, and to understand why the DLM is unable to bind the substrate, its structure was determined to 2.5 A resolution. Comparison of this structure with both wild-type ddeltac1 and ddeltac2 structures reveals that the conformations of the 20's and 70's loops in the DLM mutant are very similar to those of ddeltac2. This suggests that the five amino acid substitutions in domain 1 which lie outside of the two loop regions and which are different in the DLM, and ddeltac2, must be important enzymatically. The structure of the DLM in complex with sulfate was also determined to 2.2 A resolution. This structure demonstrates that the conformational changes of the 280's loop and domain 3, previously observed in ddeltac1, also occur in the DLM upon sulfate binding, reinforcing the hypothesis that these events may occur in the active ddeltac2 protein during catalysis.

Structural studies of duck delta2 crystallin mutants provide insight into the role of Thr161 and the 280s loop in catalysis.

Delta crystallin, a taxon-specific crystallin present in avian eye lenses, is homologous to the urea cycle enzyme ASL (argininosuccinate lyase). Although there are two delta crystallin isoforms in duck lenses, ddeltac1 (duck delta1 crystallin) and ddeltac2 (duck delta2 crystallin), only ddeltac2 is catalytically active. Previous structural studies have suggested that residues Ser283 and His162 in the multi-subunit active site of ddeltac2/ASL are the putative catalytic acid/base, while the highly conserved, positively charged Lys289 is thought to help stabilize the carbanion intermediate. The strict conservation of a small hydroxy-containing residue (Thr or Ser) at position 161 adjacent to the putative catalytic base, as well as its proximity to the substrate in the S283A ddeltac2 enzyme-substrate complex, prompted us to investigate further the role this residue. Structures of the active T161S and inactive T161D ddeltac2 mutants, as well as T161D complexed with argininosuccinate, have been determined to 2.0 A resolution. The structures suggest that a hydroxy group is required at position 161 to help correctly position the side chain of Lys289 and the fumarate moiety of the substrate. Threonine is probably favoured over serine, because the interaction of its methyl group with Leu206 would restrict its conformational flexibility. Residues larger than Thr or Ser interfere with substrate binding, supporting previous suggestions that correct positioning of the substrate's fumarate moiety is essential for catalysis to occur. The presence of the 280s loop (i.e. a loop formed by residues 270-290) in the 'open' conformation suggests that loop closure, thought to be essential for sequestration of the substrate, may be triggered by the formation of the carbanion or aci-carboxylate intermediates, whose charge distribution more closely mimics that of the sulphate ion found in the active-site region of the inactive ddeltac1. The 280s loop in ddeltac1 is in the closed conformation.

Long-term outcome of patients with urea cycle disorders and the question of neonatal screening.

UNLABELLED: With regard to the principles established for neonatal population screening, the question arises whether for patients with urea cycle disorders there is an accepted treatment which really affects the disease course and prognosis as compared to the natural history of these diseases. A retrospective study of 88 patients was performed. Based on questionnaires, the survival rate and neurodevelopmental outcome of patients treated with protein restriction alone was compared to the new therapy introduced in the 1980s with added citrulline/arginine, essential amino acids for improving the amino acid composition of the restricted natural protein and benzoate. Survival of patients with neonatal presentation was improved by the extensive therapy but this mostly at the cost of an increasing number of retarded patients. Long-term outcome did not differ significantly between the two treatments. Of all patients, 56% were symptomatic within 4 days of age and 67% within the 1st week. Thus a prevention of irreversible damage by neonatal screening on blood obtained at 3-4 days of life is questionable. Whether the benefit of obtaining a rapid diagnosis, e.g. for allowing proper counselling and prospective treatment, is acceptable for the parents of prospective patients remains open. The organisation of a dense network of specialised metabolic centres with sufficient staff and resources is a prior condition for any screening programme in order to ensure the rapid diagnosis, follow-up of treatment and counselling of a cumulative number of affected chronic patients needing this support. A commitment on a long-term basis by the institutions is needed in view of the health budget restrictions. CONCLUSION: in the short term, the goal is to detect hyperammonaemic patients as early as possible with special emphasis on sick neonates. In practice, quantitative plasma ammonia determination without delay is recommended in any newborn for whom a sepsis work-up is considered and in children who refuse feeding or vomit and show alterations of consciousness and/or neurological symptoms.

Structural studies of duck delta 1 and delta 2 crystallin suggest conformational changes occur during catalysis.

Duck delta1 and delta2 crystallin are 94% identical in amino acid sequence, and while delta2 crystallin is the duck orthologue of argininosuccinate lyase (ASL) and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate, the delta1 isoform is enzymatically inactive. The crystal structures of wild type duck delta1 and delta2 crystallin have been solved at 2.2 and 2.3 A resolution, respectively, and the refinement of the turkey delta1 crystallin has been completed. These structures have been compared with two mutant duck delta2 crystallin structures. Conformational changes were observed in two regions of the N-terminal domain with intraspecies differences between the active and inactive isoforms localized to residues 23-32 and both intra- and interspecies differences localized to the loop of residues 74-89. As the residues implicated in the catalytic mechanism of delta2/ASL are all conserved in delta1, the amino acid substitutions in these two regions are hypothesized to be critical for substrate binding. A sulfate anion was found in the active site of duck delta1 crystallin. This anion, which appears to mimic the fumarate moiety of the argininosuccinate substrate, induces a rigid body movement in domain 3 and a conformational change in the loop of residues 280-290, which together would sequester the substrate from the solvent. The duck delta1 crystallin structure suggests that Ser 281, a residue strictly conserved in all members of the superfamily, could be the catalytic acid in the delta2 crystallin/ASL enzymatic mechanism.

Role of nitric oxide in the synthesis of guanidinosuccinic acid, an activator of the N-methyl-D-aspartate receptor.

BACKGROUND: We propose that reactive oxygen and argininosuccinic acid (ASA) form guanidinosuccinic acid (GSA). An alternative to this hypothesis is the so-called guanidine cycle, which consists of a series of hydroxyurea derivatives that serve as intermediates in a pathway leading from urea to GSA. We compare the role of the guanidine cycle to that of nitric oxide (NO) in the synthesis of GSA. METHODS: The members of the guanidine cycle (hydroxyurea, hydroxylamine plus homoserine, L-canaline, and L-canavanine) were incubated with isolated rat hepatocytes. The known NO donors, NOR-2, NOC-7, and SIN-1, were incubated with ASA in vitro. Ornithine, arginine, or citrulline, which increase arginine, a precursor of NO, were incubated with isolated rat hepatocytes. GSA was determined by high-performance liquid chromatography. RESULTS: None of guanidine cycle members except for urea formed GSA. SIN-1, which generates superoxide and NO formed GSA, but other simple NO donors, did not. Both carboxy-PTIO, a scavenger of NO, and dimethyl sulfoxide, a hydroxyl radical scavenger, completely inhibited GSA synthesis by SIN-1. GSA formation by SIN-1 reached a maximum at 0.5 mmol/L and decreased at higher concentrations. GSA synthesis, stimulated by urea in isolated hepatocytes, was inhibited by ornithine, arginine, or citrulline with ammonia, but not by ornithine without ammonia, where arginine production is limited. CONCLUSION: GSA is formed from ASA and the hydroxyl radical. When arginine increased in hepatocytes, GSA synthesis decreased. These data suggest that increased NO, which results from high concentrations of arginine, or SIN-1 scavenges the hydroxyl radical. This may explain the decreased GSA synthesis in inborn errors of the urea cycle where ASA is decreased, and also the diminished GSA excretion in arginemia.

Intragenic complementation and the structure and function of argininosuccinate lyase.

Argininosuccinate lyase (ASL) catalyzes the reversible hydrolysis of argininosuccinate to arginine and fumarate, a reaction important for the detoxification of ammonia via the urea cycle and for arginine biosynthesis. ASL belongs to a superfamily of structurally related enzymes, all of which function as tetramers and catalyze similar reactions in which fumarate is one of the products. Genetic defects in the ASL gene result in the autosomal recessive disorder argininosuccinic aciduria. This disorder has considerable clinical and genetic heterogeneity and also exhibits extensive intragenic complementation. Intragenic complementation is a phenomenon that occurs when a multimeric protein is formed from subunits produced by different mutant alleles of a gene. The resulting hybrid protein exhibits greater enzymatic activity than is found in either of the homomeric mutant proteins. This review describes the structure and function of ASL and its homologue delta crystallin, the genetic defects associated with argininosuccinic aciduria and current theories regarding complementation in this protein.

p(1),p(4)-diadenosine 5'-tetraphosphate induces the uptake of arginine and citrulline by a pore on the plasma membrane of bovine aortic endothelial cells.

We have previously demonstrated that p(1),p(4)-diadenosine 5'-tetraphosphate (Ap(4)A) induces the release of nitric oxide (NO) and modulates the uptake of extracellular L-arginine (L-Arg) and L-citrulline (L-Cit) by bovine aortic endothelial cells (BAEC) [Hilderman, R.H. and Christensen, E.F. (1998) FEBS Lett. 427, 320-324 and Hilderman, R.H., Casey, T.E. and Pojoga, L.H. (2000) Arch. Biochem. Biophys. 375, 124-130]. In this communication we report that extracellular Ap(4)A enhances the uptake of L-Arg and L-Cit through a pore on the plasma membrane of BAEC that is selective for these two amino acids. We also demonstrate that Ap(2)A, which induces NO release, enhances L-Arg uptake while Ap(5)A, a vasoconstrictor, does not enhance the uptake of L-Arg. The potential physiological significance of the uptake of these two amino acids in relation to NO synthesis is discussed.

Studies of L-arginine transport in bovine aortic endothelial cells.

We have previously demonstrated that p(1),p(4)-diadenosine 5'-tetraphosphate induces the release of NO and modulates the uptake of L-arginine by bovine aortic endothelial cells (BAEC) [Hilderman, R. H., and Christensen, E. F. (1998) FEBS Lett. 407, 320-324; Hilderman, R. H., Casey, T. E., and Pojoga, L. H. (2000) Arch. Biochem. Biophys. 375, 124-130]. In this communication we characterize the uptake of L-Arg by BAEC. L-Arg is transported into BAEC by at least two different transporter systems. One transporter system is protein synthesis dependent, and L-Arg transported by this system is incorporated into proteins. The second transporter system involved in L-Arg uptake is protein synthesis independent, and uptake occurs by facilitated diffusion. The L-Arg transported by facilitated diffusion is metabolized into L-argininosuccinate. Homologous and heterologous competition uptake studies were performed using a fixed concentration of radiolabeled L-Arg, L-lysine, and L-leucine with varying concentrations of competing nonradiolabeled amino acids. The results of these competition uptake studies are consistent with the protein-synthesis-dependent uptake of L-Arg taking place through a transporter system that is highly specific for L-Arg and with the facilitated diffusion uptake taking place through a transporter that is specific for L-Arg and L-Leu.