© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
Measurements of nitric oxide concentration and hyporeactivity in rat superior mesenteric artery exposed to endotoxin
aDepartment of Pharmacology, University of Aarhus, 8000 Aarhus C, Denmark
bDepartamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain
* Corresponding author. Tel.: +45-89-421-713; fax: +45-86-128-804. Email address: us{at}farm.au.dk
Objective: The present study was designed to relate nitric oxide (NO) concentration to changes in vascular reactivity in rat superior mesenteric arteries treated with lipopolysaccharide (LPS, 10 µg ml–1, 1–8 h). Methods: In rat mesenteric arteries, isometric tension was recorded in wire myographs, protein expression was evaluated by Western blot and/or immunohistochemistry and NO concentration was measured by application of a NO specific electrode. Results: Incubation with LPS (5 h) resulted in inducible NO synthase (iNOS) expression and enhanced superoxide dismutase (Cu/Zn–SOD and Mn–SOD) expression, but it did not modify endothelial NO synthase (eNOS) expression. Noradrenaline (0.5 µM) evoked increases in NO concentration and tension by, respectively, 5.0±2.0 nM and 4.4±0.1 N m–1 (n=6). While NO concentration was unaltered, incubation with LPS reduced noradrenaline contraction to 3.5±0.2 N m–1 (n=39, P<0.05). In contrast to indomethacin, 1400 W (10 µM) restored noradrenaline contraction, while the combination of noradrenaline and oxyhaemoglobin (10 µM) evoked less contraction in LPS compared to control segments. Polyethylene glycol (PEG)-catalase in combination with oxyhaemoglobin reversed noradrenaline hyporeactivity in LPS-treated segments. LPS did not increase, but reduced basal NO concentration, an effect which was reversed by 1400 W and tempol (100 µM). In LPS-treated segments contracted with noradrenaline, the NO synthase substrate, L-arginine (100 µM), relaxed and increased NO concentration with, respectively, 73±9% and 19.5±6.5 nM (n=5). 1400 W and oxyhaemoglobin reversed L-arginine relaxation and increases in NO concentration. In contrast to tempol and PEG-catalase, N-acetylcysteine (0.1–1 mM), which is able to release NO from intracellular stores, relaxed LPS-treated tissue, an effect that was abolished by long-term, but not by short-term, incubation with 1400 W. Conclusions: The present study provides direct evidence that exposure to LPS results in induction of iNOS and SOD associated with noradrenaline hyporeactivity, while increased NO is only measured when L-arginine is present. A catalase-sensitive mechanism and release of NO from N-acetylcysteine-sensitive stores could also contribute to the vascular hyporeactivity.
KEYWORDS Arteries; Endotoxins; Nitric oxide microelectrode; Oxygen radicals; Rat superior mesenteric artery