Cloots RH, Sankaranarayanan S, de Theije CC, Poynter ME, Terwindt E, van Dijk P, Hakvoort TB, Lamers WH, Köhler SE. Ablation of Arg1 in hematopoietic cells improves respiratory function of lung parenchyma, but not that of larger airways or inflammation in asthmatic mice. Am J Physiol Lung Cell Mol Physiol 305: L364-L376, 2013. First published July 5, 2013; doi:10.1152/ajplung.00341.2012.-Asthma is a chronic inflammatory disease of the small airways, with airway hyperresponsiveness (AHR) and inflammation as hallmarks. Recent studies suggest a role for arginase in asthma pathogenesis, possibly because arginine is the substrate for both arginase and NO synthase and because NO modulates bronchial tone and inflammation. Our objective was to investigate the importance of increased pulmonary arginase 1 expression on methacholine-induced AHR and lung inflammation in a mouse model of allergic asthma. Arginase 1 expression in the lung was ablated by crossing Arg1 fl/fl with Tie2Cre tg/Ϫ mice. Mice were sensitized and then challenged with ovalbumin. Lung function was measured with the Flexivent. Adaptive changes in gene expression, chemokine and cytokine secretion, and lung histology were quantified with quantitative PCR, ELISA, and immunohistochemistry. Arg1 deficiency did not affect the allergic response in lungs and large-airway resistance, but it improved peripheral lung function (tissue elastance and resistance) and attenuated adaptive increases in mRNA expression of arginine-catabolizing enzymes Arg2 and Nos2, arginine transporters Slc7a1 and Slc7a7, chemokines Ccl2 and Ccl11, cytokines Tnfa and Ifng, mucus-associated epithelial markers Clca3 and Muc5ac, and lung content of IL-13 and CCL11. However, expression of Il4, Il5, Il10, and Il13 mRNA; lung content of IL-4, IL-5, IL-10, TNF-␣, and IFN-␥ protein; and lung pathology were not affected. Correlation analysis showed that Arg1 ablation disturbed the coordinated pulmonary response to ovalbumin challenges, suggesting arginine (metabolite) dependence of this response. Arg1 ablation in the lung improved peripheral lung function and affected arginine metabolism but had little effect on airway inflammation. airway hyperresponsiveness; arginine; inflammation ALLERGIC ASTHMA IS A CHRONIC inflammatory disorder of the lung that is characterized by a reversible limitation of the airflow due to an allergic reaction in the airways with the characteristics of a T H 2-predominant airway inflammation. The airway inflammation is initiated by the binding of inhaled allergens to complementary IgEs on IgE receptor-bearing cells. Upon activation, these cells release histamine, cytokines, and proteases, resulting in the activation of the inflammatory cascade with lung infiltration of eosinophils and mononuclear cells. Increased mucus production, mucosal edema, and a disproportionate smooth-muscle contraction lead to airway hyperresponsiveness (AHR). As a result of the recurring inflammation, airway remodeling develops as a consequence of fibrosis and airway wall thickening (2,3,6,36).Rec...

BackgroundMilk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens.ResultsBetween 23 weeks of gestation and 3 years after birth, CPS- and ASS-protein content in enterocytes was high and then declined to reach adult levels at 5 years. OAT levels declined more gradually, whereas ARG-1 was not expressed. ARG-2 expression increased neonatally to adult levels. Neurons in the enteric plexus strongly expressed ASS, OAT, NOS1 and ARG2, while varicose nerve fibers in the circular layer of the muscularis propria stained for ASS and NOS1 only. The endothelium of small arterioles expressed ASS and NOS3, while their smooth-muscle layer expressed OAT and ARG2.ConclusionThe human small intestine acquires the potential to produce arginine well before fetuses become viable outside the uterus. The perinatal human intestine therefore resembles that of rodents and pigs. Enteral ASS behaves as a typical suckling enzyme because its expression all but disappears in the putative weaning period of human infants.

Background(Over-)expression of arginase may limit local availability of arginine for nitric oxide synthesis. We investigated the significance of arginase1 (ARG1) for the development of airway hyperresponsiveness (AHR) and lung inflammation in female mice with ovalbumin (OVA)-induced allergic asthma.Methods Arg1 was ablated in the lung by crossing Arg1 fl/fl and Tie2Cre tg/− mice. OVA sensitization and challenge were conducted, and AHR to methacholine was determined using the Flexivent system. Changes in gene expression, chemokine and cytokine secretion, plasma IgE, and lung histology were quantified using RT-qPCR, ELISA, and immunohistochemistry, respectively.Results Arg1 ablation had no influence on the development of OVA-induced AHR, but attenuated OVA-induced increases in expression of Arg2 and Nos2, Slc7a1, Slc7a2, and Slc7a7 (arginine transporters), Il4, Il5 and Il13 (TH2-type cytokines), Ccl2 and Ccl11 (chemokines), Ifng (TH1-type cytokine), Clca3 and Muc5ac (goblet cell markers), and OVA-specific IgE. Pulmonary IL-10 protein content increased, but IL-4, IL-5, IL-13, TNFα and IFNγ content, and lung histopathology, were not affected. Arg1 elimination also decreased number and tightness of correlations between adaptive changes in lung function and inflammatory parameters in OVA/OVA-treated female mice. OVA/OVA-treated female mice mounted a higher OVA-IgE response than males, but the correlation between lung function and inflammation was lower. Arg1-deficient OVA/OVA-treated females differed from males in a more pronounced decline of arginine-metabolizing and -transporting genes, higher plasma arginine levels, a smaller OVA-specific IgE response, and no improvement of peripheral lung function.ConclusionComplete ablation of Arg1 in the lung affects mRNA abundance of arginine-transporting and -metabolizing genes, and pro-inflammatory genes, but not methacholine responsiveness or accumulation of inflammatory cells.Electronic supplementary materialThe online version of this article (10.1186/s12890-017-0490-7) contains supplementary material, which is available to authorized users.

Suckling mammals, including mice, differ from adults in the abundant expression of enzymes that synthesize arginine from citrulline in their enterocytes. To investigate the importance of the small-intestinal arginine synthesis for whole-body arginine production in suckling mice, we floxed exon 13 of the argininosuccinate synthetase (Ass) gene, which codes for a key enzyme in arginine biosynthesis, and specifically and completely ablated Ass in enterocytes by crossing Ass fl and Villin-Cre mice. Unexpectedly, Ass fl/fl /VilCre tg/- mice showed no developmental impairments. Amino-acid fluxes across the intestine, liver, and kidneys were calculated after determining the blood flow in the portal vein, and hepatic and renal arteries (86%, 14%, and 33%, respectively, of the transhepatic blood flow in 14-day-old mice). Relative to control mice, citrulline production in the splanchnic region of Ass fl/fl /VilCre tg/- mice doubled, while arginine production was abolished. Furthermore, the net production of arginine and most other amino acids in the liver of suckling control mice declined to naught or even changed to consumption in Ass fl/fl /VilCre tg/- mice, and had, thus, become remarkably similar to that of post-weaning wild-type mice, which no longer express arginine-biosynthesizing enzymes in their small intestine. The adaptive changes in liver function were accompanied by an increased expression of genes involved in arginine metabolism (Asl, Got1, Gpt2, Glud1, Arg1, and Arg2) and transport (Slc25a13, Slc25a15, and Slc3a2), whereas no such changes were found in the intestine. Our findings suggest that the genetic premature deletion of arginine synthesis in enterocytes causes a premature induction of the post-weaning pattern of amino-acid metabolism in the liver.

Suckling "F/A2" mice, which overexpress arginase-I in their enterocytes, develop a syndrome (hypoargininemia, reduced hair and muscle growth, impaired B-cell maturation) that resembles IGF1 deficiency. The syndrome may result from an impaired function of the GH-IGF1 axis, activation of the stresskinase GCN2, and/or blocking of the mTORC1-signaling pathway. Arginine deficiency inhibited GH secretion and decreased liver Igf1 mRNA and plasma IGF1 concentration, but did not change muscle IGF1 concentration. GH supplementation induced Igf1 mRNA synthesis, but did not restore growth, ruling out direct involvement of the GH-IGF1 axis. In C2C12 muscle cells, arginine withdrawal activated GCN2 signaling, without impacting mTORC1 signaling. In F/A2 mice, the reduction of plasma and tissue arginine concentrations to ϳ25% of wild-type values activated GCN2 signaling, but mTORC1-mediated signaling remained unaffected. Gcn2-deficient F/A2 mice suffered from hypoglycemia and died shortly after birth. Because common targets of all stress kinases (eIF2␣ phosphorylation, Chop mRNA expression) were not increased in these mice, the effects of arginine deficiency were solely mediated by GCN2.Arginine is a substrate for the synthesis of proteins, creatine, agmatine, ornithine, and nitric oxide (1). In addition, it functions as a secretagogue for hormones, such as growth hormone (GH) and insulin (2-4). Under normal conditions, endogenous arginine synthesis in adult mammals suffices to sustain daily requirements (5), but a dietary source of arginine may become necessary when demand increases under anabolic or catabolic conditions (5). For this reason, arginine is considered a conditionally essential amino acid.Arginine deficiency represents a significant metabolic problem in premature infants (6), but its cause remains unknown. In rapidly growing suckling rodents, endogenous arginine biosynthesis is crucial to compensate for the insufficient supply of arginine via the milk (7). The intestine rather than the kidney is primarily responsible for endogenous arginine synthesis in suckling piglets (8,9). In suckling rodents, the enterocytes of the small intestine also express all enzymes necessary to synthesize arginine (10 -13), while arginase is not expressed, thus allowing efficient de novo arginine biosynthesis. To examine the function of intestinal arginine synthesis, a transgenic mouse model that overexpresses arginase-I in the enterocytes of the small intestine was produced (14 -17). During the suckling period, these "F/A2" 4 mice suffer from a selective deficiency of circulating arginine (which declines to ϳ25% of controls), reduced growth of hair and skeletal muscle, and inhibition of B-cell maturation (14). The phenotype can be rescued with arginine supplementation (14), but could not be ascribed to a deficiency of arginine metabolites, such as creatine or polyamines, or to arginine-dependent ADP-ribosylation of proteins (14 -17). Furthermore, the features of the "arginine-deficiency syndrome" are not seen in mice that are deficien...