Promotion of Anti-Tuberculosis Macrophage Activity by L-Arginine in the Absence of Nitric Oxide.

Macrophages are indispensable immune cells tasked at eliminating intracellular pathogens. Mycobacterium tuberculosis (Mtb), one of the most virulent intracellular bacterial pathogens known to man, infects and resides within macrophages. While macrophages can be provoked by extracellular stimuli to inhibit and kill Mtb bacilli, these host defense mechanisms can be blocked by limiting nutritional metabolites, such as amino acids. The amino acid L-arginine has been well described to enhance immune function, especially in the context of driving macrophage nitric oxide (NO) production in mice. In this study, we aimed to establish the necessity of L-arginine on anti-Mtb macrophage function independent of NO. Utilizing an in vitro system, we identified that macrophages relied on NO for only half of their L-arginine-mediated host defenses and this L-arginine-mediated defense in the absence of NO was associated with enhanced macrophage numbers and viability. Additionally, we observed macrophage glycolysis to be driven by both L-arginine and mechanistic target of rapamycin (mTOR), and inhibition of glycolysis or mTOR reduced macrophage control of Mtb as well as macrophage number and viability in the presence of L-arginine. Our data underscore L-arginine as an essential nutrient for macrophage function, not only by fueling anti-mycobacterial NO production, but also as a central regulator of macrophage metabolism and additional host defense mechanisms.

Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

Induction of Nitric-Oxide Metabolism in Enterocytes Alleviates Colitis and Inflammation-Associated Colon Cancer.

Nitric oxide (NO) plays an established role in numerous physiological and pathological processes, but the specific cellular sources of NO in disease pathogenesis remain unclear, preventing the implementation of NO-related therapy. Argininosuccinate lyase (ASL) is the only enzyme able to produce arginine, the substrate for NO generation by nitric oxide synthase (NOS) isoforms. Here, we generated cell-specific conditional ASL knockout mice in combination with genetic and chemical colitis models. We demonstrate that NO derived from enterocytes alleviates colitis by decreasing macrophage infiltration and tissue damage, whereas immune cell-derived NO is associated with macrophage activation, resulting in increased severity of inflammation. We find that induction of endogenous NO production by enterocytes with supplements that upregulate ASL expression and complement its substrates results in improved epithelial integrity and alleviation of colitis and of inflammation-associated colon cancer.

Citrulline and arginine utility in treating nitric oxide deficiency in mitochondrial disorders.

Mitochondrial diseases arise as a result of dysfunction of the respiratory chain, leading to inadequate ATP production required to meet the energy needs of various organs. On the other hand, nitric oxide (NO) deficiency can occur in mitochondrial diseases and potentially play major roles in the pathogenesis of several complications including stroke-like episodes, myopathy, diabetes, and lactic acidosis. NO deficiency in mitochondrial disorders can result from multiple factors including decreased NO production due to endothelial dysfunction, NO sequestration by cytochrome c oxidase, NO shunting into reactive nitrogen species formation, and decreased availability of the NO precursors arginine and citrulline. Arginine and citrulline supplementation can result in increased NO production and hence potentially have therapeutic effects on NO deficiency-related manifestations of mitochondrial diseases. Citrulline is a more efficient NO donor than arginine as it results in a greater increase in de novo arginine synthesis, which plays a major role in driving NO production. This concept is supported by the observation that the three enzymes responsible for recycling citrulline to NO (argininosuccinate synthase and lyase, and nitric oxide synthase) function as a complex that can result in compartmentalizing NO synthesis and channeling citrulline efficiently to NO synthesis. Clinical research evaluating the effect of arginine and citrulline in mitochondrial diseases is limited to uncontrolled open label studies demonstrating that arginine administration to subjects with MELAS syndrome results in improvement in the clinical symptoms associated with stroke-like episodes and a decrease in the frequency and severity of these episodes. Therefore, controlled clinical studies of the effects of arginine or citrulline supplementation on different aspects of mitochondrial diseases are needed to explore the potential therapeutic effects of these NO donors.

Optimizing therapy for argininosuccinic aciduria.

Argininosuccinic aciduria (ASA) is a urea cycle disorder with a complex phenotype. In spite of a lower risk for recurrent hyperammonemic episodes as compared to the proximal disorders of ureagenesis, subjects with ASA are at risk for long-term complications including, poor neurocognitive outcome, hepatic disease and systemic hypertension. These complications can occur in spite of current standard therapy that includes dietary modifications and arginine supplementation suggesting that the presently available therapy is suboptimal. In this article, we discuss the natural history of ASA and the recent mechanistic insights from animal studies that have shown the requirement of argininosuccinate lyase, the enzyme deficient in ASA, for systemic nitric oxide production. These findings may have therapeutic implications and may help optimize therapy in ASA.

Activated microglia cells express argininosuccinate synthetase and argininosuccinate lyase in the rat brain after transient ischemia.

Argininosuccinate-synthetase (ASS), argininosuccinate-lyase (ASL) and nitric oxide synthase (NOS) act in the l-arginine-NO-l-citrulline cycle. In the rat brain, ASS is expressed in neurons, ASL in neurons and astroglia in the striatum, both are co-expressed with nNOS in medium-sized neurons. Microglia cells express iNOS and ASS after activation but no information is available on ASL and on ASS/ASL/iNOS co-expression in this glial population. The present aim was to ascertain, by immunohistochemistry, whether the microglia cells of the rat striatum and fronto-parietal cortex express ASL and ASS in control conditions and after transient ischemia induced by middle cerebral artery occlusion, and whether ASL and ASS are co-expressed with iNOS. The study was conducted 24, 72 and 144 h after reperfusion in two groups of ischemic rats with different tissue damage and survival. ASS and ASL are not expressed by microglia cells in controls while are present in most of the activated microglia cells in the ischemic rats. In those animals with longer survival, ASS and ASL were no more detectable at 144 h, while, in the animals with shorter survival, they were co-expressed with iNOS, but only at 72 h. In the cortex, at variance with the striatum, almost all of nNOS-positive neurons co-expressed ASS and ASL. In conclusion, only activated microglia cells express ASS and ASL, this expression precedes that of iNOS and does not necessarily imply its appearance. Therefore, local factors such as the NO produced by nNOS/ASS/ASL-positive neurons, could influence ASS/ASL-positive microglia cells avoiding or allowing the induction, in these cells, of iNOS.

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.

Induction of molecular chaperones in carbon tetrachloride-treated rat liver: implications in protection against liver damage.

Carbon tetrachloride (CCl4) induces liver damage, apparently through the formation of free-radical metabolites. Molecular chaperones such as heat shock protein (Hsp) of 70 kDa have been found to protect cells from various stresses. We previously found that cytosolic chaperone pairs of the Hsp70 family and their DnaJ homolog cochaperones prevent nitric oxide-mediated apoptosis and heat-induced cell death. Expression of cytosolic chaperones, including Hsp70; heat shock cognate (Hsc) 70; and DnaJ homologs dj1 (DjB1/Hsp40/hdj-1), dj2 (DjA1/HSDJ/hdj-2), dj3 (DjA2), and dj4 (DjA4), in the liver of CCl4-treated rats was analyzed. Messenger ribonucleic acids for all these chaperones were markedly induced 3-12 hours after CCl4 treatment with a maximum at 6 hours. Hsp70 and dj1 proteins were markedly induced at 6-24 hours with a maximum at 12 hours, whereas dj2 and dj4 were moderately induced at around 12 hours. Hsc70 was weakly induced after treatment, and dj3 was little induced. To better understand the significance of the induction of chaperones, the effect of preinduction of chaperones on CCl4-induced liver damage was analyzed. When chaperones were preinduced in the liver by heat treatment, increase in serum alanine aminotransferase activity after CCl4 treatment was significantly attenuated. Hsp90, another major cytosolic chaperone, also was induced by heat treatment. On the other hand, Mn- and Cu/Zn-superoxide dismutase were not induced by heat treatment or by CCl4 treatment. These results suggest that cytosolic chaperones of Hsp70 and DnaJ families or Hsp90 (or both) are induced in CCl4-treated rat liver to protect the hepatocytes from the damage being inflicted.

[Nitrogen detoxification in artificially-fed zinc-deficient rats]. / Stickstoffdetoxifikation bei künstlich ernähtren Zn-Mangelratten.

The aim of this investigation was to determine if the hyperammonaemia shown in previous zinc-deficiency experiments was the result of disturbed enzyme activities for urea synthesis caused by zinc deficiency per se or was a secondary effect of the reduced feed intake accompanying energy and protein deficiency. For this, 24 male Sprague-Dawley rats with an average body weight of 109 g were divided into two groups of 12 animals each. Both groups were force fed by intragastric tube four times daily over 11 experimental days. Group 1 received a zinc-deficient diet (1.3 mg Zn/kg diet) in a total amount of 11.6 g/day/animal. Group 2 received the zinc sulphate-supplemented control diet (25 mg Zn/kg diet) in the same amount. This technique made it possible to supply even the zinc-deficient rats with sufficient nutrients over the whole experimental period in the same manner as for the control rats, at the same time and with the same dietary amounts. At the end of the experiment, the serum zinc concentration and the alkaline phosphatase activity were significantly reduced in the zinc-deficient rats by 59 and 37%, respectively, in comparison with control animals. This showed a severe alimentary zinc-deficiency status of the animals. The concentrations of ammonia and urea, as well as the activity of glutamate dehydrogenase in serum, were not influenced by the zinc-deficient nutrition within the experimental time. Likewise, the mitochondrial activities of glutamate dehydrogenase and carbamoylphosphate synthetase in the liver were not affected by the alimentary zinc concentration. On the contrary, the activities of ornithine carbamoyltransferase and cytosolic liver enzymes argininosuccinate synthetase, argininosuccinase and arginase were significantly increased in comparison with control rats. In the case of a sufficient supply of nutrients, alimentary zinc deficiency did not cause hyperammonaemia owing to disturbed urea synthesis, as previously hypothesized.

Protective role of the L-arginine-nitric oxide synthase pathway on preservation injury after rat liver transplantation.

BACKGROUND: A major problem complicating liver transplantation is the preservation injury that results from cold storage and subsequent ischemia/reperfusion injury after organ revascularization. The L-arginine-nitric oxide (NO) pathway has been recognized to play critical roles during infection, inflammation, organ injury, and transplant rejection. Recent data indicates that NO synthesis has beneficial effects in several models of liver injury. The purpose of this study is to examine the role of the L-arginine-NO pathway on preservation injury in an experimental model of rat liver transplantation. METHODS: Orthotopic liver transplantation was performed in syngeneic (LEW to LEW) rats. Liver preservation injury was determined by measuring serum liver function tests 6 to 48 hours after transplantation. In some experiments, rats received L-arginine supplementation 0 to 24 hours after transplantation. In other experiments, NO synthase inhibitors (L-NAME or L-NIL) were injected at the time of isograft revascularization. RESULTS: L-Arginine supplementation decreased hepatic transaminase levels at all time points examined (6-48 hours). L-Arginine produced a significant improvement in liver preservation injury by 12 hours after reperfusion. The NO synthase inhibitor L-NAME caused a significant increase in liver injury 24 hours after injection. The inducible NO synthase (iNOS)-specific inhibitor L-NIL had no significant effect on liver injury. CONCLUSIONS: The results show that L-arginine supplementation and NO synthesis improve hepatic injury and have a protective role in the transplanted liver graft. The protective effect may be mediated by low-level cNOS-derived NO.

Induced biochemical and physiological changes in young and adult growing rats fed on a vegetable or animal protein diet.

The influence of diets containing faba bean or casein as sources of protein were studied in rats at two stages of development. A significant impairment of growth rate, carcass, liver and skeletal muscle were found in young and adult rats fed for a period of 10 days on raw legume. Urinary urea output and the activities of three amino acid degrading enzymes: arginase, alanine aminotransferase and arginine succinate synthetase were all affected by the dietary protein and the stage of development. Urinary creatinine excretion was higher in the adult rats, while serum cholesterol was slightly increased in the young ones. Changes in plasma zinc may be attributed to a reduced zinc bioavailability to rats from the faba bean diet. Other biochemical parameters measured (glucose, triglycerides and plasma proteins) remained unchanged in all the experimental groups. Liver DNA and RNA content (mg/g tissue) decreased with age in both dietary groups, which were accompanied by an increase in tissue size. Furthermore, liver RNA concentration (primarily a measure of protein synthesis capacity) was enhanced in the adult legume fed rats. In this context, it is suggested that other organs (particularly muscle, with lower amino acid requirements for protein synthesis as a consequence of the stunting of growth) could contribute to increase the amino acid supply to liver in the animals fed on the faba bean diet.

Investigations on the origin and metabolism of the carbon skeleton of ornithine, arginine and proline in selected animals.

The origin and metabolism of the carbon skeletons of the amino acids ornithine and arginine have been investigated in selected animals--an earthworm, an edible mollusc, a starfish, a sea-squirt, a freshwater crustacean and a rat. Only in the rat and microorganisms of sea water was any evidence obtained for the conversion of glutamate (or N-acetylglutamate) to ornithine. Apart from the crustacean, the other animals were able to synthesise the amidine moiety of arginine. All animals were able to hydrolyse (arginase) the amidine moiety from arginine and had the enzymic capacity to convert ornithine to proline. All the animals had some enzymic ability to oxidise proline to pyrroline-5-carboxylic acid. The crustacean (Cherax destructor) was able to conserve the high concentrations of arginine in its tail muscles during fasting. The hypothesis is put forward that, as arginine appears to be an essential amino acid in the diet of this animal, its demonstrated cannibalism is, among other things, a way of supplementing dietary arginine. The results are discussed in relation to the evolution of different phosphagens derived from arginine.

Regulation of enzymes involved in ornithine/arginine metabolism in the parasitic trypanosomatid Herpetomonas samuelpessoai.

The genetic regulation of enzymes involved in arginine and ornithine synthesis has been investigated in the parasitic trypanosomatid Herpetomonas samuelpessoai. The activities of two enzymes involved in arginine synthesis, ornithine carbamoyltransferase (OCTase) and argininosuccinate lyase (ASLase) were depressed whereas the enzyme citrulline hydrolase (CHase), which is involved in ornithine synthesis, was increased in arginine supplemented cultures of the parasites. The depression of OCTase activity in the presence of arginine was not due to feedback inhibition and CHase activity of uninduced cultures was not enhanced by exogeneous arginine. Studies of the kinetics of OCTase induction and repression revealed that arginine blocks OCTase synthesis but does not cause destruction of the enzyme. Ornithine, but not citrulline was found to counteract the arginine mediated repression of OCTase. Two classes of canavanine resistant mutants of H. samuelpessoai were isolated. One class was defective in arginine uptake whereas the other was affected in regulation of OCTase and ASLase which appear to be under coordinate control in H. samuelpessoai.

Effect of protein level and supplemental lysine on growth and urea cycle enzyme activity in the pig.

Two experiments were conducted with young, growing pigs to evaluate the effects of protein levels and supplemental lysine on growth, body characteristics and urea cycle enzyme activities. In the first experiment, 8-week-old pigs weighing 17 kg were fed two levels of protein (12 or 20%) until they reached slaughter weight of approximately 95 kg. In the second experiment, three levels of protein (12, 18 or 24%) and one level of additional lysine (9 g/kg of diet added to the 12% protein diet) were fed for the same interval. After slaughter, urea cycle enzyme activities (argininosuccinate synthetase, EC 6.3.4.5; argininosuccinate lyase, EC 4.3.2.1; and arginase, EC 3.5.3.1) were determined in liver homogenates. Early growth of pigs was improved by increasing the protein level (12%) but not by adding the limiting amino acid lysine to the 12% diet. The cross sectional area of the Longissimus dorsi muscle at slaughter was increased by protein level (12%) or by adding lysine to the 12% protein diet. In contrast, subcutaneous fat was decreased only by increasing the dietary protein level. The activities of arginase, argininosuccinate synthetase and argininosuccinate lyase were increased in a linear fashion by increasing the dietary protein level. In contrast, adding lysine to the 12% protein diet did not increase urea cycle enzyme activities.

Continuous culture of Reuber hepatoma cells in serum-free arginine-, glutamine- and tyrosine-deprived chemically defined medium.

The rat hepatoma cell line, H4-II-E, was grown serially over a 1-year period and about 30 passages in arginine-, glutamine-, and tyrosine-deprived and ornithine-supplemented Eagle's minimum essential medium with no supplements other than biotin. The adapted cell line, R-Y121B, proliferates in the above mentioned medium with a doubling time of about 4 days and maintains hepatic "marker" enzymes such as tyrosine aminotransferase, phenylalanine hydroxylase, and all the enzymes of the urea cycle.

Mild variant of argininosuccinic aciduria.

A 7 and one half-year-old boy with a massive excretion of argininosuccinic acid is described. He exhibited only moderate mental retardation, cerebellar ataxia and both abnormal hair and skin. Argininosuccinate lyase activity in the erythrocytes of his parents and his sister was in the range expected for heterozygotes. The patient was put on a low protein diet with arginine supplementation and improved clinically and biochemically on this regime. The variability of the phenotypic expression of argininosuccinate lyase deficiency is stressed.

Urea cycle enzyme activity in lambs fed different nitrogen levels at low dietary intakes.

Eleven Southdown male lambs averaging 19.8 kg were randomly allotted to two groups and fed diets containing 7.7% (low-N) or 15.8% (high-N) crude protein. All of the supplemental nitrogen in the high-N diet was supplied as urea. Intake of the low-N and high-N diets averaged 372.3 g and 340.5 g/day, respectively. Findings at the end of the thirty-day trial were: (1) mean body weights unchanged for the two groups; (2) plasma urea nitrogen three-fold higher in the high-N (19.07 mg/100 ml) than the low-N (6.57) animals; (3) similar hepatic activity levels of three urea cycle enzymes (ornithine transcarbamylase, argininosuccinase, arginase) in the two groups, and (4) similar liver weights and liver protein concentration. The absence of adaptive change in enzyme levels suggests the hypothesis that addition of non-protein nitrogen to maintenance diets may cause ammonia intoxication by exceeding the liver's reserve capacity for urea synthesis.

Amino acid metabolism in the chronically uremic rat.

The chronically uremic rat has been used as a model to study amino acid metabolism in uremia. Uremic rats fed low protein diets (6% casein) survived longer than uremic rats receiving either higher levels of dietary protein or a low protein diet supplemented with a mixture of nonessential amino acids. Alterations in plasma amino acid levels were observed in the uremic rats and were similar to those found in patients with renal failure. Plasma concentrations of citrulline, free tryptophan, glycine and the methylhistidines were increased and levels of serine, ornithine, lysine, total tryptophan, tyrosine, and the tyrosine-phenylalanine ratio were reduced. The metabolic basis of the altered tyrosine-phenylalanine ratio in plasma was studied. Tyrosine aminotransferase (TAT) and phenylalanine hydroxylase (PHL) activity were normal in the liver, but renal PHL activity of was decreased as compared to control rats. Tissue concentrations of citrulline were also found to be raised in liver and muscle of uremic rats. The activity of ornithine transcarbamoylase, was reduced in the liver and arginine synthetase activity was decreased in the kidneys of uremic rats. Thus elevated citrulline levels in uremic tissue appear to be caused by a decrease conversion of citrulline to arginine in the kidney. Preliminary studies of tryptophan metabolism in uremic rats have shown elevated brain levels of 5-hydroxyindoleacetic acid and increased hepatic tryptophan oxygenase activity. Increased plasma amine levels were associated with altered activities of monoamine oxidase and diamine oxidase in kidney and other tissues.

The effect of electrical stimulation of the hypothalamus and other brain areas on urea cycle enzymatic activities in the liver in rabbits--experimental studies on brain and liver interrelationship--.

Changes in urea cycle enzymatic activities in the liver induced by intracerebral electrical stimulation in rabbits with chronically implanted electrodes in the hypothalamus, the thalamus, and the limbic area were studied in comparison with these activities in non-stimulated controls. In stimulating the ventromedial nucleus of the hypothalamus, the two-hour sessions were followed by significantly reduced activity in arginase and the arginine synthetase system in the liver, but the six-hour stimulation sessions were followed by normal range activity in these enzymes. In stimulating the lateral hypothalamic area, the two-hour sessions were followed by only slightly reduced activity in the arginine synthetase system in the liver. Stimulation of the center median nucleus of the thalamus, the medial area of the amygdala and the dorsal hippocampus produced no significant changes in urea cycle enzymatic activities in the liver. The possibility that ammonia metabolism in the liver may be modified through some functional linkage between the brain and the liver was suggested.