Microcirculatory Function during Endotoxemia-A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation.

Karolina A P Wijnands,Hans M H Van Eijk,Wouter H Lamers,Peter Brouckaert,Benjamin Vandendriessche,Dennis M Meesters,Jacob J Briedé,Martijn Poeze,Anje Cauwels

doi:10.3390/ijms222111940

Abstract

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.

Highlights

Plasma citrulline concentrations were similar in all mouse lines under both basal and endotoxemic conditions, except for a higher concentration under basal conditions in Nos3−/− mice (Figure 1B)
The results presented in this study demonstrate that Nos3 deficiency does not decrease jejunal tissue nitric oxide (NO) production, but does result in an impaired microcirculation as judged from the decreased number of perfused vessels under basal and endotoxemic conditions
Our findings further reveal that, in the absence of Nos3, endotoxemia and supplementation of L-citrulline does not affect the microcirculation, demonstrating that the positive effects of L-citrulline supplementation depend on the presence of a functional NOS3 enzyme

Summary

Introduction

Multiple organ failure and mortality [1,2,3,4] These alterations in the microcirculation, if not reversed within the first 24 h after the onset of sepsis (prolonged sepsis), are hypothesized to be the single independent factor in predicting patient outcome [4]. The impaired NO production in the microcirculation is thought to be mediated by dysfunction of NOS3 [14], which is mediated at least partly by arginine deficiency [9,15,16] This arginine deficiency, in turn, results from an enhanced utilization of arginine upon the pro-inflammatory upregulation of NOS2 and arginase [15,17,18], and from a decreased import of arginine and/or an impaired resynthesis of arginine from citrulline [15,19,20,21,22]

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Results

Conclusion