Relationship between NO synthesis, arginine transport, and intracellular arginine levels in vascular smooth muscle cells

Summary. The present study was designed to evaluate the relevance of arginine transport in nitric oxide (NO) synthesis in vascular smooth muscle cells. For this purpose, NO synthesis and arginine transport (system B^0,+ and y⁺) were evaluated in cells treated with IL-1β or angiotensin II (Ang II). In addition, the effects of 5 mM lysine and glutamine, competitive inhibitors of systems y⁺ and B^0,+ respectively, were examined. L-arginine transport was estimated with ³H-labelled arginine and NO was determined with the Griess reagent. These studies were done in control conditions, arginine-starved cells, and in cells incubated in media containing 10 mM arginine. Our data indicate that induction of NO biosynthesis by IL-1β depends on external arginine when cells are arginine-depleted for 24 hours. The concentration of arginine producing half maximal activation of NO synthesis in arginine-depleted cells ([arginine]_i < 10 μM) was 41.1 ± 18 μM. By contrast, in normal culture conditions, NO synthesis occurred independently of arginine transport. Neither 5 mM lysine or glutamine which abolished arginine transport through systems y⁺ and B^0,+, respectively, reduced nitrite release in cells incubated in normal media. This suggests that the relevance of arginine uptake to NO synthesis depends on the status of intracellular arginine pools. Intracellular arginine concentrations were not affected by the stimulation of NO production using IL-1β or its inhibition using Ang II, but were markedly reduced by arginine starvation for 48 h. Aspartate levels were also reduced by arginine-depletion, but were not affected in cells incubated with 10 mM arginine. By contrast, glutamate levels were reduced in arginine-starved cells and were increased in cells incubated in arginine-supplemented medium. Ornithine levels were markedly increased by arginine supplementation. Altogether, these findings indicate that NO synthesis is normally independent of membrane transport. However in arginine-depleted cells, membrane transport is essential for NO synthesis. It is concluded that arginine transport is required for the long-term maintenance of intracellular arginine pools. Received February 7, 1999; Accepted June 21, 1999     

Synthesis and biological activity of canavanine hydrazide derivatives

Summary. The canavanine derivatives L-canavanine hydrazide (CH), L-canavanine-bis-(2-chloroethyl)hydrazide (CBCH) and L-canavanine phenylhydrazide (CPH) were synthesized and evaluated for biological activity in microorganisms, plants and tumor cells using canavanine as a positive control. (1) In microbial systems, the compounds exerted activity, as assessed in 14 bacterial strains. The effect of canavanine was easily removed by equimolar concentrations of arginine or ornithine, while the effect of CBCH or CPH was abolished by 10-fold excess of arginine or 10- to 100-fold excess of ornithine. (2) In plants, the activity of CH and CBCH were relatively low, whereas the inhibitory potential of CPH was comparable or even superior to that of canavanine, resulting at 1 mM concentration in a nearly complete block of tomato cell growth, and reducing by up to 80% the length of radicles of cress, amaranth, cabbage and pumpkin. (3) In pumpkin seeds, CPH or canavanine induced the synthesis of four small heat shock proteins of hsp-17 family in the pH range of 6 to 7.5. The proteins exhibited in both cases a similar profile, but differed in the timing of their expression and/or accumulation. With canavanine, the highest hsp-17 expression was found after 48 h of drug treatment, while with CPH this maximum was shifted to 24 h. (4) CPH proved to be highly cytotoxic against Friend leukemia cells in culture, exceeding by one order of magnitude the cytotoxicity of canavanine. The effect of canavanine was completely removed in the presence of equimolar amounts of arginine, while a 20-fold excess of arginine failed to abolish the cytotoxicity of CPH. Thus, a proper hydrazide modification of canavanine may lead to a significant increase in its growth-inhibitory activity and to a change in the mode of action of the parent compound. Received October 5, 1999, Accepted January 27, 1999     

Na+-independent Lysine Transport in Human Intestinal Caco-2 Cells

The nature of transepithelial and cellular transport of the dibasic amino acid lysine in human intestinal epithelial Caco-2 cells has been characterized. Intracellular accumulation of lysine across both the apical and basolateral membranes consists of a Na⁺-independent, membrane potential-sensitive uptake. Na⁺-independent lysine uptake at the basolateral membrane exceeds that at the apical membrane. Lysine uptake consists of both saturable and nonsaturable components. Na⁺-independent lysine uptake at both membranes is inhibited by lysine, arginine, alanine, histidine, methionine, leucine, cystine, cysteine and homoserine. In contrast, proline and taurine are without inhibitory effects at both membranes. Fractional Na⁺-independent lysine efflux from preloaded epithelial layers is greater at the basolateral membrane and shows trans-stimulation across both epithelial borders by lysine, arginine, alanine, histidine, methionine, and leucine but not proline and taurine. Na⁺-independent lysine influx (10 μm) in the presence of 10 mm homoserine shows further concentration dependent inhibition by lysine. Taken together, these data are consistent with lysine transport being mediated by systems b^o,+, y⁺ and a component of very low affinity (nonsaturable) at both membranes. The relative contribution to lysine uptake at each membrane surface (at 10 μm lysine), normalized to total apical uptake (100%), is apical b^o,+ (47%), y⁺ (27%) and the nonsaturable component (26%), and basal b^o,+ (446%), y⁺ (276%) and the nonsaturable component (20%). Northern analysis shows hybridization of Caco-2 poly(A)⁺RNA with a human rBAT cDNA probe. Received: 3 July 1995/Revised: 6 February 1996     

Liquid chromatographic—mass spectrometric analysis for screening of patients with cystinuria, and identification of cystine stone

Analyses of amino acids in the urine of a normal human and of patients with heterozygous and homozygous cystinuria have been carried out, using liquid chromatography—mass spectrometry with an atmospheric pressure ionization interface system. A kidney cystine stone was also analysed by this system. Very intense quasi-molecular ions ([M + H]⁺) of standard cystine, arginine, lysine and ornithine were observed on mass chromatograms as base peaks. Mass chromatograms of the urine samples from a normal human and from patients with heterozygous and homozygous cystinuria were easily distinguishable. The retention times in the mass chromatogram and mass spectrum of kidney stone cystine was almost the same as that of authentic cystine.     

Nitric oxide and polyamine pathway-dependent modulation of neutrophil free amino- and α-keto acid profiles or host defense capability

Summary. We have examined the effects of N_ω-nitro-L-arginine-methylester-hydrochloride [L-NAME; inhibitor of nitric oxide synthase], S-nitroso-N-acetyl-penicillamine [SNAP; nitric oxide donor], α-difluoro-methyl-ornithine [DFMO; inhibitor of ornithine decarboxylase] arginine or ornithine as well as the combination of arginine or ornithine with L-NAME, SNAP or DFMO on intracellular free amino- and α-keto acid profiles and the immune function markers superoxide anion and hydrogen peroxide generation as well as released myeloperoxidase activity in neutrophils (PMN). Although the underlying mechanisms still remain unclear, we believe from our results that nitric oxide as well as polyamine-dependent pathways are involved in the signal transmission of free radical molecule, beneficial nutritional therapy or maleficient pharmacological stress-induced alterations in PMN nutrient composition. Relevant changes in intragranulocyte free amino- and α-keto acid homeostasis and metabolism, especially, may be one of the determinants in PMN nutrition that positively or negatively influences and modulate neutrophil host defence capability and immunocompetence.     

Effects of hypoxia on plasma amino acids of fetal sheep

Summary. Secondary amino acid disturbances from circulatory responses during hypoxia may cause problems in interpreting plasma amino acid profiles of sick babies investigated for possible inherited defects. Systematic studies to characterise them are difficult in man. We investigated the effects of hypoxia on plasma amino acids by studying 9 late gestation fetal sheep in utero during 11 one hour episodes of moderately severe isocapnic hypoxia. In 6 experiments, maternal plasma amino acids were also monitored. Fourteen fetal plasma amino acids increased significantly, with the largest proportionate changes in alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, ornithine and lysine. Maternal amino acids did not increase. Probable explanations were reflex peripheral vasoconstriction in skeletal muscle beds and decreased hepatic blood flow. The findings extend our knowledge of the fetal response to hypoxic stress, demonstrate the importance of skeletal muscle in branched-chain amino acid metabolism, and should help with interpretation of postnatal plasma amino acid disturbances. Received January 29, 1999, Accepted February 22, 1999     

Decarboxylation of ornithine and lysine in rat tissues.

The possibility that arginine and lysine might be decarboxylated by rat tissues was investigated. No evidence for decarboxylation of arginine could be found. Lysine decarbosylase (L-lysine carboxy-lyase, EC 4.1.1.18) activity producing CO2 and cadaverine was detected in extracts from rat ventral prostate, androgen-stimulated mouse kidney, regenerating rat liver and livers from rats pretreated with thioacetamide. These tissues all have high ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activities. Lysine and ornithine decarboxylase activities were lost to similar extents on inhibition of protein synthesis by cycloheximide and on exposure to alpha-difluoromethylornithine. A highly purified ornithine decarboxylase preparation was able to decarboxylate lysine and the ratio of ornithine to lysine decarboxylase activities was constant throughout purification. Kinetic studies of the purified preparation showed that the V for ornithine was about 4-fold greater than for lysine, but the Km for lysine (9 mM) was 100-times greater than that for ornithine (0.09 mM). These experiments indicate that all of the detectable lysine decarboxylase activity in rat and mouse tissues was due to the action of ornithine decarboxylase and that significant cadaverine production in vivo would occur only when ornithine decarboxylase activity is high and lysine concentrations substantially exceed those of ornithine.     

Variation in proton affinity of the guanidino group between free and blocked arginine

Summary. In this paper, the analog of arginine residues in peptides was synthesized and characterized by ESI-MS/MS (electrospray ionization with tandem mass spectrometry), ³¹P NMR, ¹H NMR, IR and high-resolution mass spectrometry. When the Todd reaction activity of the guanidino group in free arginine and the arginine peptide analog were compared, it was found that the proton affinity of the guanidino group was decreased when both the N- and the C-terminal were blocked. As a result, the guanidino group of arginine residues in peptides could be phosphorylated under the Todd reaction condition, but not the free arginine. This result was further proved by the theoretical calculation of their proton affinity.     

Consequences of renal mass reduction on amino acid and biogenic amine levels in nephrectomized mice

Summary. Amino acid and biogenic amine changes were investigated in nephrectomized mice ten days postsurgery. Uremic mice exhibited changes in amino acid concentrations in plasma, urine and brain. Particularly plasma methionine, citrulline and arginine levels were significantly enhanced in nephrectomized mice compared to controls whereas serine was decreased. Urinary excretion of methionine, citrulline and alanine was higher in nephrectomized mice compared to controls whereas many amino acids were increased in brain of nephrectomized mice. Brain and urinary amino acid changes were more pronounced in the 75% than in the 50% nephrectomized mice. Brain norepinephrine and dopamine and its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were significantly increased whereas serotonin was decreased comparing the 75% nephrectomized mice to the sham-operated mice. This study demonstrates that at very early stages of renal insufficiency, specific amino acid and biogenic amine changes occur in plasma, urine and brain. These alterations might depend qualitatively and quantitatively on the degree of functional renal mass reduction. Received April 5, 1999     

Plasma arginine correlations in trauma and sepsis

Summary. Arginine (ARG) is an amino acid (AA) with unique properties and with a key-role in the metabolic, immune and reparative response to trauma and sepsis. This study has been performed to characterize the correlations between plasma levels of ARG, of other AA and of multiple metabolic variables in trauma and sepsis. Two-hundred and sixty-three plasma amino-acidograms with a large series of additional biochemical and blood variables were obtained consecutively in 9 trauma patients who developed sepsis, undergoing total parenteral nutrition with dextrose, fat and a mixed AA solution containing 10.4% arginine. ARG was low soon after trauma, then it increased with increasing distance from trauma and with the development of sepsis. ARG was also directly related to the AA infusion rate (AAIR) and for any given AAIR, was lower after trauma than after the development of sepsis. ARG was also related directly to the plasma levels of most of the other AA, the best correlation being that with lysine (r² = 0.81, p < 0.001). These correlations were often shifted downwards (showing lower ARG for any given level of the other AA) in measurements performed after trauma, compared to those performed after development of sepsis; this effect was more pronounced for the correlations with branched chain AA. Correlations between ARG and non-AA variables were not particularly relevant. The best simultaneous correlates of ARG, among variables involved in plasma ARG availability, were citrulline level, AAIR and urinary 3-methylhistidine excretion (accounting for the effect of endogenous proteolysis) (multiple r² = 0.70, p < 0.001). Plasma ornithine (ORN), the AA more specifically linked to ARG metabolism, correlated with AAIR better than ARG and, for any given AAIR, was lower after trauma than after the development of sepsis. Correlations of ORN with other AA levels were poorer than those found for ARG, however ORN was directly related to white blood cell and platelet count, fibrinogen, transferrin, cholesterol and many AA clearances. These data show that changes in ARG in trauma and sepsis are correlated with changes in other AA and, within these correlations, reconfirm a tendency to lower ARG in trauma compared to sepsis. The strong correlation with lysine warrants a deeper assessment of the practical implications of interdependency between these two AA. The data also suggest that changes in plasma ORN in trauma and sepsis may reflect adequacy of AA substrate to support acute-phase and other synthetic processes.     

l-[35S]CYSTINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— The uptake of [³⁵S]cystine at 37°C by synaptosomal fractions isolated from adult rat cerebrum can be divided into two components. About 60% of the uptake is due to binding to synaptosomal proteins while the remainder exists as a free amino acid pool. Chemical analysis of this soluble component indicates that considerable reduction of cystine to cysteine occurs with 75% or more of the labeled molecular species being cysteine. The process involved in the uptake into the soluble pool was composed of two saturable systems with apparent K _m values of 0.14 and 1.4 m m . The low K _m system was sodium and oxygen independent but inhibited by dinitrophenol. Dibasic amino acids, lysine, arginine and ornithine, did not inhibit cystine uptake. The characteristics of cystine uptake by synaptosomes from newborn brain are very similar to those of adult brain.     

Synthesis of metabolism-resistant substrates for the transport system for cationic amino acids; their stimulation of the release of insulin and glucagon, and of the urinary loss of amino acids related to cystinuria

Several amino acids have been synthesized as model transport substrates building on the piperidine and cyclohexane rings. Only when the distal N atom is part of an unambiguously cationic structure are these compounds transported predominantly by the cationic amino acid system. These amino acids in labeled form are excreted rather slowly in unmodified state, very little ¹⁴CO₂ being released. Those which are unambiguously cationic (including also homoarginine) led to a greatly increased excretion of arginine, lysine, ornithine and citrulline. Those which might be expected to act as lysine analogs had little effect on the excretion of the basic amino acids, although the excretion of citrulline and the sum of glutamine plus asparagine was accelerated. Certain of the analogs intensified the excretion of citrulline in dissociation from effects on resorption of the basic amino acids, also in dissociation from effects on cystine resorption. These results indicate citrulline resorption does not occur principally by the same agency serving for the basic amino acids, nor by the agency serving for cystine, despite the observed interactions for resorption. The injection of either of three transport analogs for arginine into the rat leads to early increases in the circulating levels of immunologically reactive insulin and glucagon.     

Almost all about citrulline in mammals

Summary. Citrulline (Cit, C₆H₁₃N₃O₃), which is a ubiquitous amino acid in mammals, is strongly related to arginine. Citrulline metabolism in mammals is divided into two fields: free citrulline and citrullinated proteins. Free citrulline metabolism involves three key enzymes: NO synthase (NOS) and ornithine carbamoyltransferase (OCT) which produce citrulline, and argininosuccinate synthetase (ASS) that converts it into argininosuccinate. The tissue distribution of these enzymes distinguishes three “orthogonal” metabolic pathways for citrulline. Firstly, in the liver, citrulline is locally synthesized by OCT and metabolized by ASS for urea production. Secondly, in most of the tissues producing NO, citrulline is recycled into arginine via ASS to increase arginine availability for NO production. Thirdly, citrulline is synthesized in the gut from glutamine (with OCT), released into the blood and converted back into arginine in the kidneys (by ASS); in this pathway, circulating citrulline is in fact a masked form of arginine to avoid liver captation. Each of these pathways has related pathologies and, even more interestingly, citrulline could potentially be used to monitor or treat some of these pathologies. Citrulline has long been administered in the treatment of inherited urea cycle disorders, and recent studies suggest that citrulline may be used to control the production of NO. Recently, citrulline was demonstrated as a potentially useful marker of short bowel function in a wide range of pathologies. One of the most promising research directions deals with the administration of citrulline as a more efficient alternative to arginine, especially against underlying splanchnic sequestration of amino acids. Protein citrullination results from post-translational modification of arginine; that occurs mainly in keratinization-related proteins and myelins, and insufficiencies in this citrullination occur in some auto-immune diseases such as rheumatoid arthritis, psoriasis or multiple sclerosis.     

Protein-bound advanced glycation endproducts (AGEs) as bioactive amino acid derivatives in foods

Summary. The Maillard reaction or nonenzymatic browning is of outstanding importance for the formation of flavour and colour of heated foods. Corresponding reactions, also referred to as “glycation”, are known from biological systems, where the formation of advanced glycation endproducts (AGEs) shall play an important pathophysiological role in diabetes and uremia. In this review, pathways leading to the formation of individual protein-bound lysine and arginine derivatives in foods are described and nutritional consequences resulting from this posttranslational modifications of food proteins are discussed.     

In silico analysis of SLC3A1 and SLC7A9 mutations in Iranian patients with Cystinuria

Molecular Biology Reports - Cystinuria is an autosomal recessive defect in reabsorptive transport of cystine and the dibasic amino acids ornithine, arginine, and lysine from renal tubule and small...     

Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures

Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (¹⁵NH₄)₂SO₄ and K¹⁵NO₃. By using two-dimensional ¹⁵N-¹H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of ¹⁵N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional ¹⁵N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid γ-aminobutyric acid in the one-dimensional ¹⁵N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into γ-aminobutyric acid, N-acetylputrescine. The occurrence of ¹⁵N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen. Received: 29 March 1999 / Accepted: 15 July 1999     

MgATP-induced inhibition of the adenosine triphosphatase activity of submitochondrial particles.

Uptake of L-cystine by brush-border membrane vesicles isolated from rat renal-cortical tissue was time-dependent and occurred in the absence of cystine reduction. A significant capacity for vesicular binding of cystine was observed. The amount bound increased with time of incubation and could be displaced by thiol reagents. At early time points, cystine uptake measured the transport of cystine into the intravesicular space. Total cystine uptake was mediated by multiple transport systems, including a low-Km high-affinity component which was shared by lysine, arginine, ornithine and glutamine and on which hetero-exchange diffusion of lysine and cystine was demonstrated.     

l-amino acid oxidases of Proteus rettgeri.

Proteus rettgeri has been found to contain two separable 1-amino acid oxidases. Both enzymes are particulate in nature, neither being ribosomal bound. One of these enzymes appears to have broad specificity, being active toward monoaminomonocarboxylic, imino, aromatic, sulfur-containing, and beta-hydroxyamino acids. The other enzyme has more limited specificity, catalyzing the oxidative deamination of the basic amino acids and citrulline. The affinity of this oxidase for the various substrates at pH 7.6 in decreasing order is arginine, histidine, ornithine, citrulline, and lysine. This enzyme has a particularly high affinity for arginine (Km equal to 0.27 mM), and anomalous kinetics are observed with increasing substrate concentrations. When concentrations of arginine greater than 1.0mM were added to the reaction containing histidine, imidazole pyruvate formation was completely inhibited.     

Isolation and Characterization of Strains Defective in Vacuolar Ornithine Permease inNeurospora crassa

Vacuolar uptake of ornithine and lysine was characterized inNeurospora crassausing a cupric ion permeabilization system. Michaelis constants were measured as 1.4 mM for lysine and 11.0 mM for ornithine, and maximal velocities were determined. Vacuolar lysine uptake was shown to be inhibited competitively byl-arginine and histidine while ornithine uptake was inhibited by a variety of amino acids. Strains defective in the vacuolar ornithine permease were isolated using a filtration enrichment method. Two isolates—RSC-39 and RSC-63—had a reduced ability to accumulate ornithine. Vacuolar uptake of amino acids was measured using cupric ion-permeabilized mycelia; both strains had reduced ornithine uptake while lysine uptake and arginine uptake were normal. For both isolates, both the Michaelis constant and the maximal velocity for ornithine uptake were reduced compared to those of wild type. These results suggest that both strains are defective in the gene which encodes the vacuolar ornithine permease.     

Polyamine-deficient Neurospora crassa mutants and synthesis of cadaverine.

The polyamine path of Neurospora crassa originates with the decarboxylation of ornithine to form putrescine (1,4-diaminobutane). Putrescine acquires one or two aminopropyl groups to form spermidine or spermine, respectively. We isolated an ornithine decarboxylase-deficient mutant and showed the mutation to be allelic with two previously isolated polyamine-requiring mutants. We here name the locus spe-1. The three spe-1 mutants form little or no polyamines and grow well on medium supplemented with putrescine, spermidine, or spermine. Cadaverine (1,5-diaminopentane), a putrescine analog, supports very slow growth of spe-1 mutants. An arginase-deficient mutant (aga) can be deprived of ornithine by growth in the presence of arginine, because arginine feedback inhibits ornithine synthesis. Like spe-1 cultures, the ornithine-deprived aga culture failed to make the normal polyamines. However, unlike spe-1 cultures, it had highly derepressed ornithine decarboxylase activity and contained cadaverine and aminopropylcadaverine (a spermidine analog), especially when lysine was added to cells. Moreover, the ornithine-deprived aga culture was capable of indefinite growth. It is likely that the continued growth is due to the presence of cadaverine and its derivatives and that ornithine decarboxylase is responsible for cadaverine synthesis from lysine. In keeping with this, an inefficient lysine decarboxylase activity (Km greater than 20 mM) was detectable in N. crassa. It varied in constant ratio with ornithine decarboxylase activity and was wholly absent in the spe-1 mutants.