Regeneration Of Arginine Research Articles

Pegylated arginine deiminase depletes plasma arginine but maintains tissue arginine availability in young pigs.

Pegylated arginine deiminase (ADI-PEG20) results in the depletion of arginine with the production of isomolar amounts of citrulline. This citrulline has the potential to be utilized by the citrulline recycling pathway regenerating arginine and sustaining tissue arginine availability. The goal of this research was to test the hypothesis that ADI-PEG20 depletes circulating arginine in pigs but maintains tissue arginine concentration and function, and to characterize the kinetics of citrulline and arginine. Two multitracer approaches (bolus dose and primed-continuous infusion) were used to investigate the metabolism of arginine and citrulline in Control (n = 7) and ADI-PEG20 treated (n = 8) pigs during the postprandial period. In addition, blood pressure was monitored by telemetry, and multiple tissues were collected to determine arginine concentration. Plasma arginine was depleted immediately after ADI-PEG20 administration, with an increase in plasma citrulline concentration (P < 0.01). The depletion of arginine did not affect (P > 0.10) blood pressure, whole body protein synthesis, or urea production. Despite the lack of circulating arginine in ADI-PEG20-treated pigs, most tissues were able to maintain concentrations similar (P > 0.10) to those in Control animals. The kinetics of citrulline and arginine indicated the high citrulline turnover and regeneration of arginine through the citrulline recycling pathway. ADI-PEG20 administration resulted in an absolute and almost instantaneous depletion of circulating arginine, thus reducing global availability without affecting cardiovascular parameters and protein metabolism. The citrulline produced from the deimination of arginine was in turn utilized by the citrulline recycling pathway restoring local tissue arginine availability.NEW & NOTEWORTHY Pegylated arginine deiminase depletes circulating arginine, but the citrulline generated is utilized by multiple tissues to regenerate arginine and sustain local arginine availability. Preempting the arginine depletion that occurs as result of sepsis and trauma with arginine deiminase offers the possibility of maintaining tissue arginine availability despite negligible plasma arginine concentrations.

Fumarase Overexpression Abolishes Hypertension Attributable to endothelial NO synthase Haploinsufficiency in Dahl Salt-Sensitive Rats.

Human blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through an arginine regeneration pathway. We examined the role of the fumarase-NO pathway in the development of hypertension using genetically engineered rat models. Dahl salt-sensitive (SS) rats with heterozygous mutation of eNOS (endothelial NO synthase or Nos3; SS-Nos3+/-) were bred with SS rats with a hemizygous Fh transgene. SS-Nos3+/- rats without the Fh transgene (SS-Nos3+/-/Fh0/0) developed substantial hypertension with a mean arterial pressure of 134.2±3.7 mm Hg on a 0.4% NaCl diet and 178.0±3.5 mm Hg after 14 days on a 4% NaCl diet. Mean arterial pressure decreased remarkably to 123.1±1.4 mm Hg on 0.4% NaCl, and 143.3±1.5 mm Hg on 4% NaCl in SS-Nos3+/- rats with a Fh transgene (SS-Nos3+/-/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS-Nos3+/-/Fh0/1 rats compared with SS-Nos3+/-/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression in rats on the 0.4% NaCl diet. However, the decrease in NO metabolite in the renal outer medulla of SS-Nos3+/-/Fh0/0 rats on the 0.4% NaCl diet was reversed in SS-Nos3+/-/Fh0/1 rats, and levels of L-arginine, but not the other 12 amino acids analyzed, were significantly higher in SS-Nos3+/-/Fh0/1 rats than in SS-Nos3+/+/Fh0/0 rats. In conclusion, fumarase has potent effects in restoring NO production and blunting the development of hypertension attributable to eNOS haploinsufficiency.

Modulation of arginine metabolic pathways as the potential anti-tumor mechanism of recombinant arginine deiminase

Arginine deiminase (ADI), currently in clinical trials, has various biological activities including anti-proliferation, anti-angiogenesis and inhibition of inducible nitric oxide synthase (iNOS). To recognize limitations and therapeutic applications, the mechanism of ADI modulation of arginine metabolic pathways was investigated. MCF-7 and A549 cells have notable different sensitivity to recombinant ADI (rADI) and express diverse argininosuccinate synthase (AS) activity, which regenerates arginine. Due to compartmentalization of arginine, utilization of arginine for protein synthesis occurs from either the intracellular arginine pool or the citrulline–arginine-regeneration pathway, whereas for polyamine synthesis, utilization is only from the intracellular arginine pool. Modulating AS activity or introducing rADI intracellularly to reduce intracellular arginine regeneration may expand therapeutic applications of rADI.

Accessibility of endothelial and inducible nitric oxide synthase to the intracellular citrulline–arginine regeneration pathway

This study investigates our hypothesis that argininosuccinate synthase (AS), the rate-limiting enzyme for arginine ( l-arg) regeneration from citrulline ( l-cit), plays a pivotal role in supplying l-arg to endothelial (eNOS), but not inducible (iNOS) nitric oxide synthase, for nitric oxide (NO) production. Transgenic rat blood–brain barrier (TR-BBB) endothelial cells were used as a model to elucidate the accessibility of the l-arg compartments for NOS isozymes. NO production via eNOS or iNOS, with or without α-methyl- dl-aspartic acid (MDLA), an AS inhibitor, was measured by a fluorometric method. NO production via eNOS was activated by the calcium ionophore A23187, while via iNOS was induced by cytokines. AS activity was assayed by the amount of argininosuccinate regenerated from radioactive aspartic acid from cell extracts. Upon increased AS activity (5.9-fold) in cells grown in l-arg-free/ l-cit-supplemented medium, A23187-activated NO production also significantly increased, however cytokine-induced NO production was not detected. A23187-activated NO production was observed not only in l-arg containing medium, but also l-arg-free and l-arg-free/ l-cit-supplemented medium, and was abolished by MDLA regardless of medium type. Cytokine-induced NO production was only observed in l-arg containing medium, not in l-arg-free or l-arg-free/ l-cit-supplemented medium, and it was not inhibited by MDLA in the l-arg containing medium. Our results indicate that extracellular l-arg was the only l-arg pool for cytokine-induced NO production and intracellular l-arg regenerated from l-cit via AS pathway was the major l-arg pool for A23187-activated NO production in TR-BBB endothelial cells. Therefore, modulation of AS activity could be a promising strategy to selectively alter NO production via eNOS, but not iNOS.

Argininosuccinate Synthase Expression Is Required to Maintain Nitric Oxide Production and Cell Viability in Aortic Endothelial Cells

Although cellular levels of arginine greatly exceed the apparent K(m) for endothelial nitric-oxide synthase, current evidence suggests that the bulk of this arginine may not be available for nitric oxide (NO) production. We propose that arginine regeneration, that is the recycling of citrulline back to arginine, defines the essential source of arginine for NO production. To support this proposal, RNA interference analysis was used to selectively reduce the expression of argininosuccinate synthase (AS), because the only known metabolic role for AS in endothelial cells is in the regeneration of l-arginine from l-citrulline. Western blot analysis demonstrated a significant and dose-dependent reduction of AS protein as a result of AS small interfering RNA treatment with a corresponding diminished capacity to produce basal or stimulated levels of NO, despite saturating levels of arginine in the medium. Unanticipated, however, was the finding that the viability of AS small interfering RNA-treated endothelial cells was significantly decreased when compared with control cells. Trypan blue exclusion analysis suggested that the loss of viability was not because of necrosis. Two indicators, reduced expression of Bcl-2 and an increase in caspase activity, which correlated directly with reduced expression of AS, suggested that the loss of viability was because of apoptosis. The exposure of cells to an NO donor prevented apoptosis associated with reduced AS expression. Overall, these results demonstrate the essential role of AS for endothelial NO production and cell viability.

The caveolar nitric oxide synthase/arginine regeneration system for NO production in endothelial cells.

The enzyme endothelial nitric oxide synthase (eNOS) catalyzes the conversion of arginine, oxygen and NADPH to NO and citrulline. Previous results suggest an efficient, compartmentalized system for recycling of citrulline to arginine utilized for NO production. In support of this hypothesis, the recycling enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase (AL), have been shown to colocalize with eNOS in caveolae, a subcompartment of the plasma membrane. Under unstimulated conditions, the degree of recycling is minimal. Upon stimulation of NO production by bradykinin, however, recycling is co-stimulated to the extent that more than 80% of the citrulline produced is recycled to arginine. These results suggest an efficient caveolar recycling complex that supports the receptor-mediated stimulation of endothelial NO production. To investigate the molecular basis for the unique location and function of endothelial AS and AL, endothelial AS mRNA was compared with liver AS mRNA. No differences were found in the coding region of the mRNA species, but significant differences were found in the 5'-untranslated region (5'-UTR). The results of these studies suggest that sequence in the endothelial AS-encoding gene, represented by position -92 nt to -43 nt from the translation start site in the extended AS mRNA 5'-UTRs, plays an important role in differential and tissue-specific expression. Overall, a strong evidential case has been developed supporting the proposal that arginine availability, governed by a caveolar-localized arginine regeneration system, plays a key role in receptor-mediated endothelial NO production.

Caveolar Localization of Arginine Regeneration Enzymes, Argininosuccinate Synthase, and Lyase, with Endothelial Nitric Oxide Synthase

Although normal intracellular levels of arginine are well above the Km, and should be sufficient to saturate nitric oxide synthase in vascular endothelial cells, nitric oxide production can, nonetheless, be stimulated by exogenous arginine. This phenomenon, termed the “arginine paradox,” has suggested the existence of a separate pool of arginine directed to nitric oxide synthesis. In this study, we demonstrate that exogenous citrulline was as effective as exogenous arginine in stimulating nitric oxide production and that citrulline in the presence of excess intracellular and extracellular arginine further enhanced bradykinin stimulated endothelial nitric oxide production. The enhancement of nitric oxide production by exogenous citrulline could therefore be attributed to the capacity of vascular endothelial cells to efficiently regenerate arginine from citrulline. However, the regeneration of arginine did not affect the bulk intracellular arginine levels. This finding not only supports the proposal for a unique pool of arginine, but also suggested channeling of substrates that would require a functional association between nitric oxide production and arginine regeneration. To support this proposal, we showed that nitric oxide synthase, and the enzymes involved in arginine regeneration, argininosuccinate synthase and argininosuccinate lyase, cofractionated with plasmalemmal caveolae, a subcompartment of the plasma membrane. Overall, the results from this study strongly support the proposal for a separate pool of arginine for nitric oxide production that is defined by the cellular colocalization of enzymes involved in nitric oxide production and the regeneration of arginine.

Isolation and characterization of glyoxal-arginine modifications.

5-(4,5-Dihydroxy-2-imino-1-imidazolidinyl)norvaline (1) was identified as the only product of the early reaction of arginine with glyoxal, which was slowly degraded to N(5)-[[(carboxymethyl)amino](imino)methyl]ornithine (3, N(7)-carboxymethylarginine). No other structures could be detected within a range of pH 4-8 and 20-50 degrees C in reaction conditions. The rates of formation for both products increased with pH and temperature. In equilibrium, the vincinal diol groups of 1 were 86% trans configured. The formation of 1 was reversible, as could be shown by cis-trans isomerization of the separated isomers and by regeneration of arginine in the presence of the alpha-dicarbonyl trapping reagents, o-phenylenediamine and aminoguanidine. Both 1 and 3 were converted to 5-(2-imino-5-oxo-1-imidazolidinyl)norvaline (2) only under strong acidic conditions.

Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle. Role in the regeneration or arginine for nitric oxide synthesis

Nitric oxide synthase produces NO, citrulline, water, and NADP at the expense of arginine, NADPH, and dioxygen. While citrulline has been considered to be an inert by-product of the high output inducible isoform of NO synthase (iNOS), we show here that immunostimulants induce a metabolic pathway in vascular smooth muscle cells, which enables them to regenerate arginine from citrulline. Regeneration of arginine from citrulline is accomplished by two urea cycle enzymes: arginino-succinate synthetase (AS) and argininosuccinate lyase (AL). Whereas AL is constitutive to vascular smooth muscle cells, AS mRNA and enzyme activity is markedly induced in cells by treatment with bacterial lipopolysaccharide (LPS). The induction of AS mRNA and activity by LPS follows a time course which mirrors that for iNOS but lags 1-2 h behind. As shown for iNOS, interferon-gamma does not itself induce AS but is synergistic with LPS. AS induction is suppressed by glucocorticoids, actinomycin D, and, to a lesser extent, cycloheximide. On the other hand, AS induction is unaffected by an excess of citrulline or the inhibitor of iNOS, N omega-methyl-L-arginine. Our results show the urea cycle enzymes AS and AL confer cells with the capacity to produce NO without a need for exogenous arginine. In conjunction with NOS, citric acid cycle enzymes that covert fumarate to oxaloacetate (fumarase and malate dehydrogenase) and oxaloacetate to aspartate (aspartate transaminase), AS and AL form a novel arginine-citrulline cycle that enables high output NO production by cells.

Diketopinic acid — A novel reagent for the modification of arginine

Diketopinic acid has been synthesized and shown to be a reagent of choice for specific, reversible modification of the guanidine groups of arginine residues. Diketopinic acid is a yellow crystalline substance and the carboxyl group of the reagent is a convenient handle for attachment to other molecules. The adducts of diketopinoyl derivatives with the guanidine group are cleaved by 0.2 M o-phenylenediamine at pH 8–9. The modification and regeneration of arginine and of arginyl residues in soyabean trypsin inhibitor and insulin are presented as demonstrations of the use of the reagent. The use of diketopinoyl resin in the separation of oxidized A and B chains of insulin has been discussed.

Camphorquinone-10-sulfonic acid and derivatives: convenient reagents for reversible modification of arginine residues.

Camphorquinone-10-sulfonic acid hydrate was prepared by the action of selenous acid on camphor-10-sulfonic acid. Camphorquinone-10-sulfonylnorleucine was prepared either from the sulfonic acid via the sulfonyl chloride or by selenous acid oxidation of camphor-10-sulfonylnorleucine. These reagents are useful for specific, reversible modification of the guanidino groups of arginine residues. Camphorquinonesulfonic acid is a crystalline water-soluble reagent that is especially suitable for use with small arginine-containing molecules, because the sulfonic acid group of the reagent is a convenient handle for analytical and preparative separation of products. Camphorquinonesulfonylnorleucine is more useful for work with large polypeptides and proteins, because hydrolysates of modified proteins may be analyzed for norleucine to determine the extent of arginine modification. The adducts of the camphorquinone derivatives with the guanidino group are stable to 0.5 M hydroxylamine solutions at pH 7, the recommended conditions for cleavage of the corresponding cyclohexanedione adducts. At pH 8-9 the adducts of the camphorquinone derivatives with the guanidino group are cleaved by o-phenylenediamine. The modification and regeneration of arginine, of the dipeptide arginylaspartic acid, of ribonuclease S-peptide, and of soybean trypsin inhibitor are presented as demonstrations of the use of the reagents. The use of camphorquinonesulfonyl chloride to prepare polymers containing arginine-specific ligands is discussed.