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Cardiovascular Research 2004 62(1):202-211; doi:10.1016/j.cardiores.2004.01.014
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Measurements of nitric oxide concentration and hyporeactivity in rat superior mesenteric artery exposed to endotoxin

Raquel Hernanza,b, María J Alonsob, Helle Zibrandtsena, Yolanda Alvarezb, Mercedes Salaicesb and Ulf Simonsen*,a

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

Received 7 August 2003; revised 8 January 2004; accepted 9 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
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


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Endotoxic shock is characterized by hypotension, vascular collapse and multiple organ failure [1]. That nitric oxide (NO) plays a pivotal role in these effects rests on the observation of increased activity and expression of inducible NO synthase (iNOS) in a wide variety of tissues, including the vascular wall [2–4], and of increased plasma levels of the NO end products, nitrite and nitrate [5]. Administration of NO synthase inhibitors reduces plasma nitrite and nitrate levels [6,7] and increases vascular tone and blood pressure in patients with septic shock [8]. Moreover, iNOS-deficient mice do not present hyporeactivity to vasoconstrictor stimuli [9], and were suggested to exhibit less hypotension and mortality after lipopolysaccharide (LPS) than wild-type controls [10]. Thus, the evidence appears incontrovertible that NO participates in the hypotension associated with vascular collapse in endotoxic shock; however, evidence for a direct relationship between iNOS-derived NO and vascular relaxation is lacking.

At least two mechanisms can explain the lack of a direct relationship between NO concentration and relaxation in vascular preparations treated with endotoxin. Sepsis also results in a large increase of superoxide anion possibly by induction of vascular xanthine oxidase and NADPH oxidase [11], although it is possible that iNOS and cyclooxygenase-2 (COX-2) also contribute to generation of radical oxygen species (ROS) [12]. An increase in both NO and superoxide anion leads to formation of peroxynitrite [13], effectively reducing the biovailability of NO. In spite of elevated superoxide anion levels, it was shown that iNOS activity could lead to the formation of releasable NO stores [14], which may facilitate the removal of excessive NO. The main candidates for these stores are protein-bound dinitrosyl–iron complexes (DNIC) [14] and high-molecular-weight S-nitrosothiols [15]. Low-molecular-weight thiols such as N-acetylcysteine are able to displace NO from these species by release of NO [16]. Both increased superoxide production and/or formation of NO stores would result in measurements of low NO concentrations compared to hyporeactivity after LPS.

The aim of the present study was to gain evidence for a direct relationship between iNOS-derived NO and vascular hyporeactivity. For this purpose, a polarographic NO-selective electrode was introduced into the lumen of the mounted arterial segment, allowing simultaneous measurements of variations in NO concentration and force. The arterial segment was treated with the bacterial wall component, LPS, and the role of the presence of the substrate L-arginine and the NO stores formation in the reduced noradrenaline vasoconstriction was addressed. Moreover, the preparations were examined for expression of endothelial NO synthase (eNOS), iNOS, superoxide dismutase (Cu/Zn–SOD, Mn–SOD) and COX-2, and indomethacin was applied to elucidate whether prostaglandins contribute to noradrenaline hyporeactivity.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Mechanical recordings
Adult male Wistar rats (12–16 weeks old) were killed by cervical dislocation and exsanguinated by decapitation. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications No. 85-23, revised 1996). Segments from the proximal part between aorta and the first branch of the superior mesenteric artery were dissected in cold (4 °C) physiological salt solution (PSS) [17] and mounted on 100-µm wires in a small vessel myograph for isometric tension recording as previously described [17]. Contractility was evaluated by adding high K+ solution (125 mM), which was PSS with NaCl exchanged for KCl on equivalent basis.

Vascular segments were incubated with either vehicle or LPS (10 µg ml–1, 1–8 h) and contraction to noradrenaline (0.5 µM) was measured each hour. In noradrenaline-contracted preparations, concentration–response curves were constructed for L-arginine (0.01 µM–0.1 mM) before and after 3 and 5 h, N-acetylcysteine (0.1–1 mM) before and after 7 h, acetylcholine (1 nM–10 µM) before and after 1 and 6 h, and for exogenously generated NO (10 nM–10 µM) 8 h after LPS treatment.

To evaluate the role of iNOS-derived NO and COX-derived prostanoids in the effect of LPS, segments were incubated, respectively, with the specific iNOS inhibitor, 1400 W (10 µM) and with the COX inhibitor, indomethacin (10 µM) during the entire experiment (long-term incubation). To rule out the role of NO stores, 1400 W was added only 30 min before addition of noradrenaline followed by N-acetylcysteine after 7 h LPS incubation (short-term incubation). To analyse the participation of free NO, the NO scavenger, oxyhaemoglobin (10 µM), was added to arterial segments contracted with noradrenaline, 3 min before relaxation–response curves were constructed.

N-acetylcysteine has been described to have antioxidant properties [18]. Therefore, the effect of the antioxidants, polyethylene glycol (PEG)-catalase (300 U ml–1) and tempol (10 µM–1 mM), was also examined in segments contracted with noradrenaline after 7 h of incubation with or without LPS. Moreover, a series of experiments were performed where the combination of PEG-catalase and oxyhaemoglobin on noradrenaline hyporeactivity was examined. Relaxations for hydrogen peroxide (H2O2, 1 mM) were obtained in the absence and the presence of PEG-catalase.

2.2. Simultaneous measurements of NO concentration and force
For simultaneous measurement of force and NO concentration, a NO sensitive microelectrode (ISONOP30, World Precision Instruments, Stevenage, UK) with a diameter of 30–50 µm was first calibrated by use of NO solution and then introduced into the lumen of the artery mounted in the myograph as previously described [17]. In some experiments, calibration of the electrode was performed before and after the experimental protocol and the sensitivity remained unchanged (n=5 electrodes). To test selectivity of the electrodes, a lack of response to sodium nitrite up to 10 µM was taken as evidence for an intact coating of the electrode. Noradrenaline is oxidized on carbon fibres, where coating is damaged, and electrodes were discarded if noradrenaline (0.5–1 µM) in the absence of vascular tissues increased electrode current. The high concentrations of N-acetylcysteine applied in the present study increased electrode current in the absence of vascular tissue and precluded measurement of NO concentration in these experiments.

The effect of vehicle and LPS on noradrenaline contraction and acetylcholine (10 µM) relaxation, as well as in the NO increase induced by both agents, was evaluated before and after 6 h incubation. Increases in NO concentration and relaxations induced by L-arginine (30 and 100 µM) in noradrenaline-contracted segments were measured before and after 3 and 5 h incubation in the absence and the presence of LPS. Basal NO levels were estimated before and after 6 h incubation in the absence or in the presence of LPS by measuring the decrease in NO concentration induced by addition of the NO scavenger, oxyhaemoglobin, for 3 min.

2.3. Immunohistochemistry
To study the localization of iNOS, eNOS, COX-2, Cu/Zn–SOD, and Mn–SOD after LPS treatment (5 h), segments were fixed with cold (4 °C) 4% paraformaldehyde, pH 7.0, for 1 h, and embedded in paraffin. Longitudinal sections 5 µm thick were obtained and processed following the avidin–biotin–peroxidase method as previously described [19]. Thus, the sections were incubated overnight with either mouse monoclonal antibody anti-iNOS (1:200, Transduction Laboratories, Lexington, UK) or anti-eNOS (1:1000, Transduction Laboratories), rabbit polyclonal antibody anti-COX-2 (1:700, Cayman Chemical, Ann Arbor, MI, USA), anti-Cu/Zn–SOD (1:1000, StressGen, Victoria, Canada), or anti-Mn–SOD (1:4000, StressGen) diluted in 1% bovine serum albumin. Then, the sections were reacted with a biotinylated antimouse/antirabbit immunoglobulins followed by incubation with streptavidin which was conjugated to horseradish peroxidase (LSAB 2 kit for rat tissue, DAKO, Denmark), and the immunocomplex was visualized as a brown product after incubating with 0.05% 3,3-diamino-benzidine and 0.0225% H2O2. Controls were obtained using arterial sections incubated without the corresponding primary antibody.

2.4. Western blot
From each animal, two arterial segments of 3 mm length were incubated for 5 h in oxygenated PSS (37 °C) with or without LPS, quick-frozen in liquid nitrogen and kept at –70 °C until protein expression analysis. Western blots for iNOS, Cu/Zn–SOD, Mn–SOD, and COX-2 were performed as previously described [19], while for eNOS expression analysis membranes were incubated with mouse monoclonal antibody for eNOS (1:2500) followed by secondary antibodies (1:2000, Transduction Laboratories). Data for protein expression are expressed as a ratio of {alpha}-actin expression, which was determined on the same membranes by using a mouse monoclonal antibody (1:3000000, Boehringer Mannheim, Mannheim, Germany). Human endothelial cells were used as positive control for eNOS.

2.5. Materials
Drugs used were: LPS (Escherichia coli, serotype 055:B5); acetylcholine hydrochloride; noradrenaline-hydrochloride; indomethacin; L-arginine hydrochloride; N-acetyl-L-cysteine; PEG-catalase; 4-hydroxy-tempol and bovine serum albumin (Sigma, St. Louis, MO, USA); 1400 W dihydrochloride (Alexis Biochemicals, San Diego, CA, USA); SDS and acrylamide (Bio-Rad Laboratories, Hercules, CA, USA); xylene (Merck Eurolab, Denmark); paraformaldehyde (Kebo Lab, Denmark); ethanol (Aalborg, Denmark); Mayer's acid haematoxylin (Bie and Berntsen, Denmark) and 3,3-diamino-benzidine (DAKO).

Except for indomethacin, which was dissolved in NaHCO3 (0.5% w/v), stock solutions were made in bidistilled water and kept at –20 °C until use. Oxyhaemoglobin and NO solutions were prepared as previously described [17].

2.6. Calculations and statistical evaluation
Mechanical responses of the arteries are expressed as increases in force {Delta}F (m N) or as active wall tension, {Delta}T (N m–1), which is {Delta}F divided by twice the segment length. Contractions obtained in the absence and in the presence of LPS are expressed as a percentage of the noradrenaline contraction obtained in the beginning of the experiment, while vasodilator responses are expressed as a percentage of noradrenaline contraction.

Data are expressed as mean±S.E.M. of n rats. Differences between means were analysed using one-way analysis of variance (ANOVA) followed by a Bonferroni t test, unpaired or paired Student's t test, as indicated. The effect of the incubation time, as well as differences between concentration–response curves were analysed by two-way ANOVA followed by a Bonferroni t test. Probability value of less than 5% was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. NOS, SOD and COX-2 expression in LPS-treated arteries
In arterial segments incubated with LPS, iNOS immunoreactivity was induced in adventitia and the endothelium, but it was not detected in control preparations (Fig. 1a and b). Western blots confirmed increased expression of iNOS in LPS-treated segments (Fig. 2a). eNOS was present in the endothelium (results not shown), and quantification by Western blot showed eNOS protein content was similar in control and LPS-treated arteries (Fig. 2b).


Figure 1
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  		Fig. 1 Lipopolysaccharide (LPS)-induced iNOS and superoxide dismutase immunoreactivity in rat superior mesenteric artery. Immunoreactivity for iNOS was not detected in control tissue (a), but was present in adventitia and media of preparations incubated for 5 h with LPS (10 µg ml–1) (b). (c–f) Cytosolic Cu/Zn–SOD and Mn–SOD immunoreactivity was detected in all three layers but was more pronounced in sections from LPS-treated compared to control arterial segments. Positive immunoreaction is observed as a brown precipitate, while cell nuclei are stained blue with Mayer's haemotoxylin. Magnification x 20, n=5–6.

 

Figure 2
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  		Fig. 2 Lipopolysaccharide increases iNOS and superoxide dismutase protein expression. Representative Western blots and quantitative analysis for (a) iNOS, (b) eNOS, (c) Cu/Zn–SOD, and (d) Mn–SOD expression in rat superior mesenteric arteries incubated for 5 h in the absence or presence of lipopolysaccharide. *P<0.05 vs. control, paired Student t test, n=4–8.

 
Immunoreactivity for cytosolic Cu/Zn–SOD was sparsely located in all three layers of control sections, but was pronounced in endothelium and subendothelial area of LPS-treated segments (Fig. 1c and d). Immunoreactivity for Mn–SOD was mainly in the media and endothelium of control sections, and the reaction was pronounced in these layers of LPS-treated sections (Fig. 1e and f). Quantification by Western blot analysis also revealed that the expression of Cu/Zn–SOD and Mn–SOD was higher in LPS-treated that in control segments (Fig. 2c and d).

No COX-2 immunoreactivity was detected in sections of vascular segments fixed just after sacrifice of the animal, but COX-2 immunoreactivity was found in all three layers after 5 h; however, the intensity of the reaction was less pronounced in preparations incubated in the absence than in the presence of LPS (results not shown). Quantification by Western blot analysis also revealed that the expression of COX-2 was higher in LPS-treated than in control segments (results not shown).

3.2 Noradrenaline hyporeactivity and effect of L-arginine
In endothelium intact segments noradrenaline (0.5 µM) induced stable contractions (4.4±0.1 N m–1, n=39), which were maintained through the successive administrations (Fig. 3). When arterial segments were incubated with LPS, the response to noradrenaline decreased in an incubation time-dependent way from the fifth hour of LPS incubation (Fig. 3), where it was 3.5±0.2 N m–1 (P<0.05, n=39). However, LPS incubation (8 h) did not modify the contraction induced by 125 mM K+ (4.2±0.3 vs. 4.9±0.2 N m–1, n=39, P>0.05).


Figure 3
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  		Fig. 3 Lipopolysaccharide-induced reduction of noradrenaline contraction is inhibited by 1400 W and oxyhaemoglobin. (a) Effect of incubation with LPS on the contraction induced by noradrenaline (NA). Effect on NA contraction in control and LPS-treated segments of (b) indomethacin (Indo, 10 µM), (c) an iNOS inhibitor, 1400 W (10 µM), and (d) NO scavenger, oxyhaemoglobin (OxyHb, 10 µM). Results are means±S.E.M. of 7–39 preparations and are expressed as percentage of the contraction elicited by NA at 0 h in each case. *P<0.05 vs. control, #P<0.05 vs. LPS, +P<0.05 vs. control+OxyHb, ANOVA followed by Bonferroni t test.

 
In spite of COX-2 expression, the COX inhibitor, indomethacin (10 µM) did not modify the noradrenaline contraction in control and LPS-treated segments (Fig. 3b). However, long incubation with the specific iNOS inhibitor, 1400 W (10 µM), antagonised the inhibitory effect of LPS on noradrenaline contraction, but did not modify noradrenaline contraction in control segments (Fig. 3c). Similar results were observed when 1400 W was added only 30 min before noradrenaline administration at 7 h LPS incubation (short incubation, results not shown). In noradrenaline-contracted segments, oxyhaemoglobin (10 µM) caused an additional contraction that was higher in LPS-treated than in control segments, but the total contraction elicited by noradrenaline plus oxyhaemoglobin was lower in LPS-treated than in control segments (Fig. 3d).

To examine whether hyporeactivity to noradrenaline was associated with increased NO formation, measurements of NO concentration was performed. Simultaneous to contraction, noradrenaline induced an increase in NO concentration (Figs. 4 and 5a)Go, which was not modified by 5 h of LPS incubation (Fig. 5a). Furthermore, the incubation of LPS-treated segments with 10 µM 1400 W did not alter the increase in NO concentration (Fig. 5a). In segments without endothelium, the contraction induced by noradrenaline was augmented (6.4±0.7 N m–1, n=4, unpaired Student's t test, P<0.05), while the increase in NO concentration was abolished (n=4, results not shown).


Figure 4
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  		Fig. 4 L-arginine increases NO and inhibits noradrenaline contraction in lipopolysaccharide-treated segments. Original trace recordings of simultaneous measurements of increases in NO concentration (upper trace) and force (lower trace) in (A) control conditions and (B) after LPS (5 h) incubation. Segments were contracted with noradrenaline (NA, 0.5 µM) and L-arginine (L-Arg) was added. L-Arg relaxed the LPS-treated segment, but it had no effect in control conditions, where acetylcholine (ACh) relaxed the artery.

 

Figure 5
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  		Fig. 5 The iNOS inhibitor, 1400 W, blocks L-arginine-evoked increases in NO concentration and relaxation in lipopolysaccharide-treated vascular segments. Averages of simultaneous measurements of (a) increases in NO concentration and noradrenaline (0.5 µM) contraction (n=3–6), and of (b) L-arginine (100 µM)-evoked increases in NO concentration and relaxation as percentage of noradrenaline contraction in control conditions and after incubation with LPS and effect of 1400 W (10 µM) (n=4–6). *P<0.05 vs. parallel control; #P<0.05 vs. LPS-treated segments, ANOVA followed by Bonferroni t test.

 
The availability of L-arginine is a limiting step in the activity of iNOS [20]. Therefore, the effect of L-arginine on increases in NO concentration was examined. L-Arginine (0.03–0.1 mM) simultaneously relaxed and increased the NO concentration in noradrenaline-contracted segments after 3 and 5 h of incubation with LPS, but not in the absence of LPS (Figs. 4 and 5b)Go; both NO increase and relaxation after 5 h of LPS incubation were abolished by 1400 W (Fig. 5b).

3.3 Noradrenaline hyporeactivity and effect of N-acetylcysteine
N-Acetylcysteine (0.1–1 mM), which releases NO from intracellular stores, induced relaxations in 21 of 28 segments treated with LPS by 7 h and contracted with noradrenaline but not in the control segments (Fig. 6a). N-Acetylcysteine relaxation was inhibited by oxyhaemoglobin (10 µM) and by long incubation with 1400 W (10 µM) (Fig. 6a); nevertheless, short-term incubation with 1400 W did not change N-acetylcysteine relaxation (Fig. 6a).


Figure 6
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  		Fig. 6 Long-term but not short-term incubation with 1400 W blocks N-acetylcysteine-evoked relaxation in lipopolysaccharide-treated superior mesenteric artery. (a) Average N-acetylcysteine relaxations obtained in control, LPS-treated, and LPS-treated segments incubated either permanently (long) or 30 min (short) with 1400 W (10 µM), or oxyhaemoglobin (OxyHb, 10 µM). (b) Effect of tempol (100 µM), polyethylene glycol (PEG)-catalase (300 U m–1), and the combination of PEG-catalase and oxyhaemoglobin on noradrenaline contraction. Relaxations are expressed as percentage of NA contraction and are means±S.E.M. of 4–21 preparations. *P<0.05 vs. control; #P<0.05 vs. LPS-treated segments, ANOVA followed by Bonferroni t test.

 
In contrast to N-acetylcysteine, the cell permeable antioxidants, tempol (100 µM) and PEG-catalase (300 U/ml), tended to increase contraction in noradrenaline-activated LPS-treated arteries (Fig. 6b). In case of PEG-catalase, the combination with oxyhaemoglobin reversed noradrenaline hyporeactivity in LPS-treated segments (Fig. 6b). H2O2 (1 mM) relaxed noradrenaline-contracted control preparations with 83±2% (n=6), and these relaxations were abolished in the presence of PEG-catalase (n=6, results not shown).

3.4. NO concentration in LPS-treated arteries
In endothelium intact segments, the basal NO concentration, evaluated by adding the NO scavenger, oxyhaemoglobin, was not modified during the experiment in control segments. After 6 h incubation with LPS, basal NO release was not increased, but was decreased (Table 1). In LPS-treated segments 1400 W did not modify the basal NO concentration, but NO was increased by the combination of 1400 W and tempol (Table 1). In LPS-treated arteries, L-arginine (100 µM) increased the NO concentration by 24.4±3.2 nM (n=3), which was reversed with 43.8±12.0 nM (n=3) by oxyhaemoglobin.


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  		Table 1 Average changes in NO concentration ({Delta}NO, nM) and tension ({Delta}T, N m–1) induced by oxyhaemoglobin (OxyHb) and acetylcholine (ACh) in rat superior mesenteric artery in the absence (control) and in the presence of LPS, LPS+1400 W (10 µM) and LPS+1400 W+tempol (100 µM) (n=2–13)

 
LPS (10 µg ml–1) did not change acetylcholine (10 µM)-evoked increases in NO concentration and relaxation (Table 1). Thus, acetylcholine evoked relaxations with pD2-values of 7.75±0.14 and 7.56±0.17 (n=20), respectively, in control and LPS-treated segments, while exogenously added NO relaxed control segments with pD2-values and maximal relaxations of, respectively, 6.21±0.05 and 93.5±1.8% (n=6), and in segments treated with LPS with pD2-values and maximal relaxations of, respectively, 6.21±0.36 and 91.3±2.2% (n=6).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The main finding of the present study is that LPS results in induction of iNOS and SOD associated with noradrenaline hyporeactivity, which is reversed by an iNOS inhibitor, 1400 W, or by the combination of the NO scavenger, oxyhaemoglobin, and catalase. Moreover, the formation of N-acetylcysteine-sensitive stores could contribute to the vascular hyporeactivity.

4.1. Noradrenaline hyporeactivity
In human septic shock, iNOS is thought to lead to hyporeactivity to conventional vasopressors [3]. In vitro LPS was also observed to result in hyporeactivity to vasoconstrictors in rat superior mesenteric artery [21,22] and other systemic arteries [23,24]. However, LPS incubation did not modify noradrenaline contraction in mesenteric resistance arteries [2,25], and in rat aorta and cerebral arteries LPS increased 5-hydroxytryptamine contractions, although contraction to other agonists was reduced [19,24]. In the present study, LPS incubation induced iNOS expression in the vascular wall and the induction of iNOS was accompanied by decreased noradrenaline contraction, and these findings suggest iNOS-derived NO or NO containing substance play a role for LPS-evoked reduction in noradrenaline contraction. The effect of LPS appeared specific for noradrenaline because vasoconstrictions induced by high concentrations (125 mM) of potassium were not reduced. Others have previously reported treatment with LPS reduces contractions induced by potassium, but in these studies, lower concentrations of potassium (60 mM) were applied [24]. In addition to activation of soluble guanylyl cyclase, ATP-sensitive potassium channels have been demonstrated to contribute to iNOS-mediated relaxation [26]. Activation by high extracellular potassium would close the potassium channels, and hence explain why the effect of LPS is specific for noradrenaline in the present study.

Both increased endothelium-derived NO [27] and COX-2-derived prostaglandins [4] have been suggested to be involved in vascular hyporeactivity in endotoxic shock. However, eNOS expression and increases in NO concentration and relaxations evoked by acetylcholine were not increased in the present study, and makes it is unlikely that endothelium-derived NO contributes to noradrenaline hyporeactivity. Despite COX-2 expression was markedly enhanced, the lack of effect of indomethacin does not support a role for prostaglandins in the reduced noradrenaline contractions in rat mesenteric arteries. Finally, in the present study an inhibitor of iNOS, 1400 W, completely restored noradrenaline contraction, in agreement with similar observations by others in mesenteric arteries [21]. These observations support that iNOS is the main source leading to noradrenaline hyporeactivity in the rat superior mesenteric artery exposed to endotoxin.

Despite the expression of iNOS and pronounced effect on noradrenaline hyporeactivity of 1400 W, and the extracellular NO scavenger, oxyhaemoglobin, increases in NO concentration evoked by noradrenaline were not different in the LPS-treated arteries and basal NO concentration was even decreased. These results suggest that NO recorded in the lumen is not always a reflection of NO bioavailability in the vessel wall. Both binding of iNOS-derived NO in stores [14] and reaction of NO with ROS-like superoxide forming peroxynitrite [13] or with H2O2 forming hydroxyl anion (OH–) ([28], Fig. 7) could contribute to the decreased bioavailability of NO, perturbing the relationship between measured NO concentration and vascular hyporeactivity, and help to explain why treatment with oxyhaemoglobin is not associated with complete reversal of noradrenaline hyporeactivity.


Figure 7
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  		Fig. 7 Model for smooth muscle hyporeactivity to noradrenaline in arteries exposed to lipopolysaccharide. The induction of iNOS and SOD protein expression results in formation of hydrogen peroxide (H2O2), which in the presence of iron [Fe(II)] consumes NO and form nitrite (NO2–) and hydroxyl anions (OH–). Inhibition of enzymes or scavenging of NO by oxyhaemoglobin (oxyHb) is indicated with broken lines. Guanylyl cyclase (GC).

 
In the present study, basal NO release, measured by addition of oxyhaemoglobin, was decreased in LPS-treated segments. Recently, it was suggested that expression of iNOS was associated with decreased endothelium-dependent relaxation and release of NO from eNOS due to lack of L-arginine [29]. In the present study, L-arginine also restored basal NO release which provides further evidence that lack of L-arginine plays a pivotal role for decreased endothelium-derived formation of NO in LPS-treated tissue. However, L-arginine is also a scavenger of oxygen radicals [30], and the lack of this substrate for iNOS has been reported to lead to increased formation of superoxide [12]. LPS can upregulate enzymes such as NADPH oxidase capable of generating superoxide [11], and increased superoxide formation was measured in endotoxaemia [31]. In the present study, the superoxide dismutase mimetic, tempol, in combination with 1400 W, but not 1400 W alone, restored basal NO formation. Therefore, both the lack of L-arginine and increased production of superoxide anions seem to contribute to the decreased endothelium-derived basal formation of NO in LPS-treated arteries.

Production of superoxide leads to formation of H2O2 via superoxide dismutase [13], which both in case of Cu/Zn–SOD and Mn–SOD is markedly expressed in LPS-treated arteries in the present study. In the presence of iron in the smooth muscle layer [16], H2O2 reacts with NO generating nitrite and hydroxyl anions [28], which like H2O2 also leads to vasodilatation [32]. Removal of H2O2 by catalase alone will increase bioavailability of NO and that could explain why PEG-catalase by itself does not reverse noradrenaline hyporeactivity in LPS-treated arteries, but requires the presence of both oxyhaemoglobin and PEG-catalase (see also Fig. 7).

The availability of L-arginine is a limiting step in the activity of iNOS [20], and L-arginine concentrations were recently reported to be low in the splanchnic region of pigs with endotoxaemia [33]. In the present study, the administration of L-arginine in both concentration-dependent and time-dependent manners evoked relaxations of LPS-treated preparations. These findings agree with previous studies showing L-arginine evoked inhibition of contractile responses in segments from aorta and mesenteric small arteries only after LPS treatment [25,34,35]. L-Arginine relaxation was associated with significant increases in NO concentration in LPS-treated arteries of magnitudes similar to those evoked by acetylcholine. Moreover, L-arginine-evoked increases in NO concentration and relaxation were abolished by inhibition of iNOS and the NO scavenger, oxyhaemoglobin, thus providing direct evidence that iNOS-derived NO mediates L-arginine associated reduction of noradrenaline contraction in the rat superior mesenteric artery.

4.2. LPS and formation of NO stores
Based on a series of studies of vascular contractility and electron paramagnetic resonance of LPS-treated arteries, Muller et al. [16] suggested that iNOS-derived NO accumulates in stores of protein-bound dinitrosyl–iron complexes. Moreover, it was found that addition of N-acetylcysteine converts protein bound DNIC to low-molecular-weight DNIC followed by relaxation [23,36]. Low-molecular-weight DNIC can also transfer NO to other metalloproteins modifying its activity through transnitrosylation [37]. In the present study, N-acetylcysteine evoked relaxations, which were inhibited by long-term incubation with 1400 W and by oxyhaemoglobin suggesting that iNOS-derived NO mediates these relaxations. Moreover, the observation that short-term incubation with 1400 W did not inhibit N-acetylcysteine relaxation suggests that it is necessary to inhibit formation of NO stores during LPS treatment to prevent N-acetylcysteine relaxation.

4.3. Perspective
The present study provides direct evidence that exposure to LPS results in induction of iNOS associated with increased formation of NO and noradrenaline hyporeactivity, while increased NO is only measured when L-arginine is present. Both formation of ROS and N-acetylcysteine-sensitive NO stores can facilitate the removal of excessive NO, perturbing the relationship between NO concentration and vascular hyporeactivity. Further studies are necessary to explore the possibility that in addition to iNOS inhibitors, catalase mimetics may be useful means for the treatment of hypotension in endotoxic shock.


   Acknowledgements
 
We thank Heidi Knudsen for technical assistance. Ulf Simonsen was supported from the Danish Heart Foundation (99-2-2-35-227420) and the Danish Medical Research Council, while Raquel Hernanz, María J. Alonso, Yolanda Alvarez and Mercedes Salaices were supported by grants from DGICYT (SAF2003-00633) and FISS (C03/01).


   Notes
 
Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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