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Impaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia. To study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg • g bodyweight(-1) • h(-1)), combined with either L-Citrulline (6.25 mg • h-1), L-Arginine (6.25 mg • h(-1)), or L-Alanine (isonitrogenous control; 12.5 mg • h(-1)) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot. L-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level. In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

Arginase-1 is an important component of the intricate mechanism regulating arginine availability during immune responses and nitric oxide synthase (NOS) activity. In this study Arg1(fl/fl)/Tie2-Cre(tg/-) mice were developed to investigate the effect of arginase-1 related arginine depletion on NOS2- and NOS3-dependent NO production and jejunal microcirculation under resting and endotoxemic conditions, in mice lacking arginase-1 in endothelial and hematopoietic cells. Arginase-1-deficient mice as compared with control mice exhibited higher plasma arginine concentration concomitant with enhanced NO production in endothelial cells and jejunal tissue during endotoxemia. In parallel, impaired jejunal microcirculation was observed in endotoxemic conditions. Cultured bone-marrow-derived macrophages of arginase-1 deficient animals also presented a higher inflammatory response to endotoxin than control littermates. Since NOS2 competes with arginase for their common substrate arginine during endotoxemia, Nos2 deficient mice were also studied under endotoxemic conditions. As Nos2(-/-) macrophages showed an impaired inflammatory response to endotoxin compared to wild-type macrophages, NOS2 is potentially involved. A strongly reduced NO production in Arg1(fl/fl)/Tie2-Cre(tg/-) mice following infusion of the NOS2 inhibitor 1400W further implicated NOS2 in the enhanced capacity to produce NO production Arg1(fl/fl)/Tie2-Cre(tg/-) mice. Reduced arginase-1 activity in Arg1(fl/fl)/Tie2-Cre(tg/-) mice resulted in increased inflammatory response and NO production by NOS2, accompanied by a depressed microcirculatory flow during endotoxemia. Thus, arginase-1 deficiency facilitates a NOS2-mediated pro-inflammatory activity at the expense of NOS3-mediated endothelial relaxation.

With the increasing incidence of fractures now, and in the future, the absolute number of bone-healing complications such as nonunion development will also increase. Next to fracture-dependent factors such as large bone loss volumes and inadequate stabilization, the nutritional state of these patients is a major influential factor for the fracture repair process. In this review, we will focus on the influence of protein/amino acid malnutrition and its influence on fracture healing. Mainly, the arginine-citrulline-nitric oxide metabolism is of importance since it can affect fracture healing via several precursors of collagen formation, and through nitric oxide synthases it has influences on the bio-molecular inflammatory responses and the local capillary growth and circulation.

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 μg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3−/− compared to Nos2−/− mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2−/− mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3−/− or Nos2−/−/Nos3−/− mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2−/− mice), as this beneficial effect was absent in Nos3−/− or Nos2−/−/Nos3−/− mice.

Enhanced arginase-induced arginine consumption is believed to play a key role in the pathogenesis of sickle cell disease-induced end organ failure. Enhancement of arginine availability with l-arginine supplementation exhibited less consistent results; however, l-citrulline, the precursor of l-arginine, may be a promising alternative. In this study, we determined the effects of l-citrulline compared to l-arginine supplementation on arginine-nitric oxide (NO) metabolism, arginine availability and microcirculation in a murine model with acutely-enhanced arginase activity. The effects were measured in six groups of mice (n = 8 each) injected intraperitoneally with sterile saline or arginase (1000 IE/mouse) with or without being separately injected with l-citrulline or l-arginine 1 h prior to assessment of the microcirculation with side stream dark-field (SDF)-imaging or in vivo NO-production with electron spin resonance (ESR) spectroscopy. Arginase injection caused a decrease in plasma and tissue arginine concentrations. l-arginine and l-citrulline supplementation both enhanced plasma and tissue arginine concentrations in arginase-injected mice. However, only the citrulline supplementation increased NO production and improved microcirculatory flow in arginase-injected mice. In conclusion, the present study provides for the first time in vivo experimental evidence that l-citrulline, and not l-arginine supplementation, improves the end organ microcirculation during conditions with acute arginase-induced arginine deficiency by increasing the NO concentration in tissues.

To study the role and (sub) cellular nitric oxide (NO) constitution in various disease processes, its direct and specific detection in living cells and tissues is a major requirement. Several methods are available to measure the oxidation products of NO, but the detection of NO itself has proved challenging. We visualized NO production using a NO-sensitive copper-based fluorescent probe (Cu 2FL2E) and two-photon laser scanning microscopy (TPLSM). Cu 2FL2E demonstrated high sensitivity and specificity for NO synthesis, combined with low cytotoxicity. Furthermore, Cu 2FL2E showed superior sensitivity over the conventionally used Griess assay. NO specificity of Cu 2FL2E was confirmed in vitro in human coronary arterial endothelial cells and porcine aortic endothelial cells using various triggers for NO production. Using TPLSM on ex vivo mounted murine carotid artery and aorta, the applicability of the probe to image NO production in both endothelial cells and smooth muscle cells was shown. NO-production and time course was detected for multiple stimuli such as flow, acetylcholine and hydrogen peroxide and its correlation with vasodilation was demonstrated. NO-specific fluorescence and vasodilation was abrogated in the presence of NO-synthesis blocker L-NAME. Finally, the influence of carotid precontraction and vasorelaxation validated the functional properties of vessels. Specific visualization of NO production in vessels with Cu 2FL2E-TPLSM provides a valid method for studying spatial-temporal synthesis of NO in vascular biology at an unprecedented level. This approach enables investigation of the pathways involved in the complex interplay between NO and vascular (dys) function.

Fracture healing and nonunion development are influenced by a range of biological factors. Adequate amino acid concentrations, especially arginine, are known to be important during normal bone healing. We hypothesize that bone arginine availability in autologous bone marrow grafting, when using the reamer-irrigator-aspirator (RIA) procedure, is a marker of bone healing capacity in patients treated for nonunion. Seventeen patients treated for atrophic long bone nonunion by autologous bone grafting by the RIA procedure were included and divided into two groups, successful treatment of nonunion and unsuccessful, and were compared with control patients after normal fracture healing. Reamed bone marrow aspirate from a site distant to the nonunion was obtained and the amino acids and enzymes relevant to arginine metabolism were measured. Arginine and ornithine concentrations were higher in patients with successful bone healing after RIA in comparison with unsuccessful healing. Ornithine concentrations and arginase-1 expression were lower in all nonunion patients compared to control patients, while citrulline concentrations were increased. Nitric oxide synthase 2 (Nos2) expression was significantly increased in all RIA-treated patients, and higher in patients with a successful outcome when compared with an unsuccessful outcome. The results indicate an influence of the arginine–nitric oxide metabolism in collected bone marrow, on the outcome of nonunion treatment, with indications for a prolonged inflammatory response in patients with unsuccessful bone grafting therapy. The determination of arginine concentrations and Nos2 expression could be used as a predictor for the successful treatment of autologous bone grafting in nonunion treatment.

Arginine, a semi-essential amino acid is an important initiator of the immune response. Arginine serves as a precursor in several metabolic pathways in different organs. In the immune response, arginine metabolism and availability is determined by the nitric oxide synthases and the arginase enzymes, which convert arginine into nitric oxide (NO) and ornithine, respectively. Limitations in arginine availability during inflammatory conditions regulate macrophages and T-lymfocyte activation. Furthermore, over the past years more evidence has been gathered which showed that arginine and citrulline deficiencies may underlie the detrimental outcome of inflammatory conditions, such as sepsis and endotoxemia. Not only does the immune response contribute to the arginine deficiency, also the impaired arginine de novo synthesis in the kidney has a key role in the eventual observed arginine deficiency. The complex interplay between the immune response and the arginine-NO metabolism is further underscored by recent data of our group. In this review we give an overview of physiological arginine and citrulline metabolism and we address the experimental and clinical studies in which the arginine-citrulline NO pathway plays an essential role in the immune response, as initiator and therapeutic target.