Specific mediator inhibition by the NO donors SNP and NCX 2057 in the peripheral lung: implications for allergen-induced bronchoconstriction

Background The aim of this study was to examine potential therapeutic effect of the two NO donors NCX 2057 (3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid) 4-(nitrooxy)butyl ester) and SNP (sodium nitroprusside) on the early allergic airway response in the peripheral lung. Methods The experiments were performed in guinea pig lung parenchyma (GPLP) derived from ovalbumin (OVA) sensitized guinea pigs. The effects of NCX 2057 and SNP were evaluated by contractile responses and mediator release during OVA challenge. The generation of nitrite and nitrate was assessed by chemiluminescence. Statistical analysis was evaluated by ANOVA. Results Cumulatively increasing concentrations of OVA (1–10,000 ng/ml) induced concentration-dependent contractions of the GPLP that were reduced by NCX 2057 (100 μM, p < 0.001) and SNP (100 μM, p < 0.05). Antigen-induced eicosanoid release was decreased by NCX 2057 (100 μM, p < 0.001) but not by SNP (100 μM), whereas the release of histamine was reduced by SNP (100 μM, p < 0.001) but not by NCX 2057 (100 μM). In addition, NCX 2057 (0.1–100 μM), but not SNP (0.1–100 μM), relaxed leukotriene D4 (10 nM) precontracted GPLP (p < 0.01). The guanylyl cyclase inhibitor ODQ had no effect on the NCX 2057 mediated relaxation. SNP released significantly less nitrite than NCX 2057. Conclusion Although both SNP and NCX 2057 reduced the release of pro-inflammatory mediators, their profiles were distinctly different. Furthermore, NCX 2057 also induced smooth muscle dilation in the GPLP. The findings point to specific anti-inflammatory effects of different NO donors in the peripheral lung tissue.


Background
Administration of exogenous nitric oxide (NO) and nitro vasodilators has received considerable attention, mainly due to their therapeutic ability and haemodynamic effects, and are well established drugs for treatment of cardiac disorders [1,2]. Exogenous NO also has the ability to exert bronchodilatory effects in bronchial asthma [3] and NO is used in the treatment of preterm children to improve lung capacity [4]. However, the effect of NO donors during the early allergic airway response requires further evaluation, especially in the distal lung.
In the peripheral lung, the release of histamine and eicosanoids (leukotrienes and prostaglandins) from activated inflammatory cells, such as mast cells and macrophages, may contribute significantly to the symptoms of allergic rhinitis and asthma [5][6][7][8]. In the airways, mast cells and alveolar macrophages also represent a major source of NO [2,[9][10][11], which may act both directly on smooth muscle cells and in an autocrine fashion to suppress allergen-induced responses, as release of histamine [12] and leukotriene synthesis [13]. Inhibitors of NO synthases have been shown to enhance antigen-induced bronchoconstriction in sensitized guinea pigs by increased generation of leukotrienes [14].
The aim of this study was therefore to examine the role of NO donors in antigen-induced responses in the peripheral part of the lung. Thus, two structurally different NO donors, NCX 2057 (3-(4-Hydroxy-3-methoxyphenyl)-2propenoic acid) 4-(nitrooxy) butyl ester) [15] and SNP (sodium nitroprusside; Na 2 [Fe(CN) 5 NO]*2H 2 0) were used ( fig 1). The substance NCX 2057 (fig 1a) belongs to a newly class of developed NO donors that are chemically conjugated to a variety of therapeutic drugs, including the anti-histamine cetirizine [15] and NSAIDs [16]. NCX 2057 has also been shown to have anti-inflammatory properties [17]. Therefore, the parent compound of NCX 2057, ferulic acid, was also studied, as this substance has been described to have anti-inflammatory potential [18]. The other NO donor, SNP (fig 1b), is a vasodilator, used in cardiovascular treatments to lower blood pressure or to improve cardiac function [19].
The experiments were performed in actively sensitized guinea pig lung parenchyma (GPLP), an in vitro model for antigen-induced contractions [20]. Recent studies of antigen-induced airway constriction in the guinea pig lung indicate that the responses to allergen in humans are similar to the responses obtained in the guinea pig [21,22], since histamine, cysteinyl-leukotrienes (CysLTs) and several prostanoids contribute to the antigen-induced airway constriction in these particular species. The hypothesis to be tested was that NO donors in the peripheral lung may affect antigen-induced contractions either generally by dilatation of smooth muscle tissue or specifically via actions on release of histamine or eicosanoids. The data revealed that the tested NO donors SNP and NCX 2057 acted as inhibitors of allergen-induced mediator release.

Animals and OVA-sensitization
Male Dunkin Hartley guinea pigs (300-350 g b.w.) were sensitized to Chicken Egg Albumin (OVA) at least four weeks prior to experiments [14]. The study was approved by the regional committee of animal experimentation ethics (N127/04).

Lung parenchymal strips and organ bath experiments
The animals were sacrificed by an overdose of inhaled CO 2 and the heart-lung-package was quickly removed and placed in ice-cold Tyrode's solution (prepared each day, containing NaCl 149.2 mM, KCl 2.7 mM, NaHCO 3 11.9 mM, glucose 5.5 mM, CaCl 2 1.8 mM, MgCl 2 0.5 mM, NaH 2 PO 4 0.4 mM). The lung parenchyma was cut parallel to the peripheral margins, yielding four to eight strips, each having a size of 2 × 2 × 20 mm and a weight of approximately 60 mg. The lung parenchymal strips were set up at a resting tension of 2.5 mN (0.25 g) in 5 ml organ baths filled with Tyrode's solution, bubbled with carbogen gas (6.5% CO 2 in O 2 ) to keep a pH of 7.4 and the temperature was kept at 37°C [14,15]. Changes in smooth muscle tension, contractions and relaxations, were recorded via isometric force-displacement transducers connected to a Grass polygraph and responses were dis-Chemical structure of the NO donors A, NCX 2057 and B, SNP Figure 1 Chemical structure of the NO donors A, NCX 2057 and B, SNP. The arrow indicates cleavage site of NO conjugation on substance NCX 2057.

ONO 2
A B played by using the IOX data acquisition system (EMKA, France). Data were analyzed by the software program Dataanalyst (EMKA, France). After an equilibration period of 90 min and washes each 15 min, histamine (1-30 μM) was added cumulatively as a control of the GPLP reactivity. Preparations displaying contraction responses less than 1.0 mN to the highest concentration of histamine were excluded from further experiments. Another wash and equilibration period between histamine and treatment period was performed. All drugs were given 15 min before the challenges.
OVA was added as cumulative challenge of increasing concentrations (1-10,000 ng/ml) every 10 min without changing bath fluid. The cumulative OVA concentrations were chosen to generate a concentration-response curve with no contractions at the lowest dose of 1 ng/ml and maximum contractions at the final dose of 10,000 ng/ml OVA. For study of smooth muscle contractions, the GPLP was contracted with cumulative doses of LTD 4 (0.1-100 nM). 10 nM of LTD4 generated a 50% contraction of the GPLP and this EC 50 dose was chosen for studies of smooth muscle relaxation. These doses have previously been used in the GPLP in vitro test system [14,15,20]. The GPLP was precontracted with LTD 4 (10 nM) before cumulative addition of cGMP analogue 8-Bromo-cGMP, SNP or NCX 2057. cGMP-dependent relaxation was evaluated by the addition of the guanylyl cyclase inhibitor ODQ (30 μM), which previously has been demonstrated to inhibit vasodilation induced by NCX 2057 [15]. Maximum contractions of the preparation were determined with histamine (1 mM), acetylcholine (1 mM) and KCl (50 mM) at the end of each experiment, and other responses were expressed as percent of maximum contractions.

Measurements of released mediators with enzyme immunoassays
A 1-mL aliquot of organ bath fluid was collected from each organ bath and immediately frozen at -20°C. The samples were taken at the end of the equilibration period to obtain basal mediator release from the tissue and at the obtained contractile plateau after challenge with OVA 1000 ng/ml or at the obtained contractile plateaus after cumulative doses of OVA (1, 10, 100 and 1000 ng/ml). Enzyme immunoassay (EIA) analyses of the different mediators LTB 4 , CysLTs and the prostanoids thromboxane (TXA 2 ) and prostaglandin (PG) D 2 were performed according to the manufacture's instructions. TXA 2 was measured as the stable metabolite TXB 2 . PGD 2 was measured as PGD 2 -mox. The assay detection limits in the bath fluid levels for the different mediators were 7.8 pg/ml for TXB 2 , PGD 2 and LTE 4 and 3.9 pg/ml for LTB 4 . Results below detection limits were set as zero in the statistical evaluation. The EIA specificity for the different mediators to interfere with each other was less than 0.01%, with the exception of the EIA kit for TXB 2 cross reacted with PGD 2 (0.53%) and with PGE 2 (0.09%). The EIA kit for CysLTs was performed with the CysLT antiserum and the sera cross reacted with LTC 4 (100%) and LTD 4 (100%) and LTE 4 (67%). Histamine was measured as previously described [23,24]. Duplicates of 300 μl were placed in 96wells plates and the amount of histamine was analyzed by a fluorospectrometer at the wavelength 450 nM. The detection limit for histamine was 3.9 ng/ml.

Measurements of nitrite and nitrate with chemiluminescence
Aliquots of bath fluid were collected from the organ bath for measurements of nitrite and nitrate to obtain an indirect assessment of the NO releasing profile for the NO donors. To determine the basal production of nitrite and nitrate in the bath fluid, a sample volume of 200 μl was withdrawn from each bath before the addition of any drug. Bath fluid aliquots (200 μl) were then collected at 15, 30, 60, 90 and 120 min after the addition of Tyrode's solution, DMSO, NCX 2057 (100 μM) or SNP (100 μM). The amount of nitrite and nitrate was analyzed with a chemiluminescence method as previously described [25].

Data analysis and statistical procedures
All data are presented as mean ± standard error of the mean (s.e.m.). Statistical analyses were made for paired and unpaired observations by Student's t-test or analyses of variances (ANOVA). All concentration-response curves were statistically analyzed with two-way ANOVA followed by the post hoc tests Tukey's t-test or Bonferroni's t-test. A p-value of less than 0.05 was considered significant.

Effect on the release of mediators upon ovalbumin challenge
The mediator release from the lung tissue was analyzed in the bath fluid to further describe the inhibitory effects of SNP and NCX 2057 on the OVA-induced contractions. Basal release in the bath fluid before OVA challenge was below detection limit for histamine, CysLTs and LTB 4 , whereas measurable levels were obtained for TXB 2 (46 ± 11 pg/ml) and PGD 2 (56 ± 16 pg/ml) (fig 3). After challenge with OVA (1000 ng/ml) measurable levels were obtained for all the mediators with the rank order of histamine >> TXB 2 > PGD 2 > CysLTs > LTB 4 3). To see any distinction in the release profile between histamine and CysLTs the bath fluid was analysed for all the cumulative doses of OVA (1-1000 ng/ml). Histamine was released at low doses of OVA and did not increase significantly at higher doses of OVA (fig 4a). In contrast, CysLTs were dose-dependently generated after cumulative administration of OVA (fig 4b). The release of histamine was significantly inhibited by SNP (100 μM, p < 0.001), whereas NCX 2057 (100 μM) did not significantly affect this release (fig 4a). The synthesis of CysLTs was significantly inhibited by NCX 2057 (100 μM, p < 0.001) after cumulative administration of OVA (fig 4b).

Analysis of nitrate and nitrite generation from SNP and NCX 2057
NCX 2057 (100 μM, n = 3) generated nitrite with a peak of 2280 ± 824 nM after 30 min. The nitrite generation was at one hour replaced by the generation of nitrate (1597 ± 345 nM) that continuously increased to 5729 ± 466 nM at two hours ( fig 5). SNP (100 μM; n = 3) continuously released nitrite at around 800 nM for two hours in the organ bath ( fig 5). The generation of nitrate from SNP was not possible to assess due to unspecific binding during analysis. The solvent DMSO (5 μl) or Tyrode's buffer did not generate any nitrite or nitrate (n = 3, fig 5).  0.001, fig 7). Lower doses of NCX 2057 had no effect on the LTD 4 -induced contractions.

Discussion
In this study we evaluated the effect of SNP and NCX 2057 during antigen-induced contractions in the peripheral lung. Our data indicated that the tested NO donors acted specifically on the release of different antigen-induced mediators and also differently as smooth muscle dilators. These data imply that depending on the nature of the NO donor, specific inhibition of antigen-induced contraction occurs and results in consistent inhibition of contraction in response to antigen.
Recent studies of antigen-induced airway constriction in the guinea pig lung indicate that histamine, CysLTs and several prostanoids, as PGD 2 and TXA 2 , contribute to the antigen-induced airway constriction [20][21][22]. Histamine is released from granulars in mast cells and basophils and histamine is an important mediator of the early allergic airway response and airway inflammation in asthma [5]. Leukotrienes are biosynthesised de novo via 5-lipoxygenase from cell membrane phospholipids in mast cells, eosinophils, neutrophils, basophils and alveolar macrophages [26]. Prostanoids are generated de novo by cyclooxygenase (COX) enzymes in most cells [7]. However, PGD 2 is mainly produced by mast cells and is known as a marker of mast cell activation [27] and associated with inflammatory conditions [28]. PGD 2 is released in the absence of degranulation, and may be stored or rapidly synthesized and released in distinct pathways from degranulation after IgE ligation. TXA 2 is mainly produced by platelets, macrophages and neutrophils [7]. TXA 2 is increased in the airways of asthmatics after allergen chal-Drug effects on the concentration-response to OVA (1-10,000 ng/ml) in sensitized lung parenchymal strips Figure 2 Drug effects on the concentration-response to OVA (1-10,000 ng/ml) in sensitized lung parenchymal strips. A, effect of pretreatment with NCX 2057 (1 μM; n = 5), NCX 2057 (10 μM; n = 6) and NCX 2057 (100 μM; n = 5) compared to control (n = 6). B, effect of pretreatment with SNP (100 μM; n = 5) compared to control (n = 10). C, effect of pretreatment with 100 μM ferulic acid (n = 7) compared to control (n = 7). Data are expressed as mean ± s.e.m.; statistical analysis was performed by two-way ANOVA. *P < 0.05; ***P < 0.001 vs control. Histamine (ng/ml) *** lenge [6]. In the present study, measurements of histamine and eicosanoids in the bath fluid after challenges with antigen showed that histamine was released in highest amounts followed by prostanoids and then leukotrienes from the GPLP preparations. However, leukotrienes are known to be 1000 times more potent bronchoconstrictors than histamine and the induced contractions are more long lasting [29]. Interestingly, NCX 2057 and SNP had different inhibitory profile on the measured mediators in the present study. SNP blocked the release of histamine after challenge with OVA, whereas NCX 2057 reduced the synthesis of leukotrienes and prostanoids, suggesting that SNP and NCX 2057 may attenuate the activation of inflammatory cells and mediator release in distinctly different steps. NOx-containing molecules may react with oxygen, superoxides, water, nucleotides, metalloproteins, thiols, amines, and lipids to form products with biochemical actions [2] in the peripheral lung tissue. NO may also bind to iron-containing enzymes via nitrosylation and form iron-nitrosyl complexes, resulting in altered enzymatic activity and function [30]. S-nitrosylation is an important posttranslational modification that regulates NO transport and cell signalling, and thereby the activity of a vast number of proteins in different cell types, including mast cells and macrophages [30][31][32], and may be a crucial regulator of airway responsiveness [33]. There are implications that NO decrease leukotriene synthesis in mast cells [13] and macrophages [11]. A possible explanation of the inhibitory effects of NCX2057 in the present study, is therefore that this lipophilic NO donor has the potential to inhibit de novo synthesis of leukotrienes and prostanoids via interaction with iron centre in 5-lipoxygenase and cyclooxygenase enzymes [34,35], and via interaction with thiol groups on LTC 4 synthase [34]. In a previous study in the GPLP in vitro system, inhibition of NO synthases enhanced antigen-induced contractions by increasing the synthesis of CysLTs in the peripheral lung [14]. The effect of NCX 2057

OVA (-log,mg/ml)
Effect of pretreatment with SNP (100 μM, n = 4, grey) and NCX 2057 (100 μM, n = 4, black) compared to control (Tyrode's solution, n = 4, white) on mediator release from sensitized GPLP after cumulative challenge with OVA Figure 4 Effect of pretreatment with SNP (100 μM, n = 4, grey) and NCX 2057 (100 μM, n = 4, black) compared to control (Tyrode's solution, n = 4, white) on mediator release from sensitized GPLP after cumulative challenge with OVA. A, Histamine (ng/ml). B, CysLTs (pg/ml). All samples were collected at baseline and then after 10 min at the plateau of cumulative addition of 1, 10, 100 and 1000 ng/ml OVA. All data are expressed as mean ± s.e.m.; statistical analysis was performed by two-way ANOVA.*P < 0.05; ***P < 0.001 vs control. on the antigen-induced response could also be explained by the fact that this molecule is derived from the parent compound ferulic acid, a substance described to have anti-inflammatory and anti-oxidant potentials [18,36]. NCX 2057 has previously been implicated to have antiinflammatory properties [17]. Nonetheless, in the present study the ferulic acid substance did not influence either the antigen-induced contractions or the release of proinflammatory mediators, suggesting that the inhibitory effect of NCX 2057 is due to its NO conjugation. The other tested NO donor, SNP, completely reduced the release of histamine after OVA challenge in this study. Topical administration of SNP has previously been shown to reduce histamine-induced plasma exudation both in guinea pig in vivo and human nasal airways [37] and previous findings in the rat mast cell confirm that both SNP and nitrite may inhibit the release of histamine [38,39], suggesting that SNP has a specific mode of action on degranulation. SNP was over all less effective than NCX 2057 in this study. Previous studies in the GPLP in vitro system show that antagonists to the histamine receptor, such as cetirizine, has very little effect on the early allergic response in the peripheral lung [15]. This is in line with  1-100 nM) in GPLP compared to control (n = 9). All data are expressed as mean ± s.e.m.; statistical analysis was performed by two-way ANOVA. ***P < 0.001 vs control. SNP did not relax the lung parenchyma whereas higher concentrations of NCX 2057 (100 μM) induced relaxation of the smooth muscle tissue in the present study. These results are in contrast to the previous findings in guinea pig aorta and pulmonary artery where NCX 2057 induced vasodilation at much lower concentrations (nM) [15], supporting the hypothesis that the smooth muscle tissue in the peripheral lung is less sensitive to NO mediated dilations. The guanylyl cyclase inhibitor ODQ did not inhibit the relaxation induced by NCX 2057 despite the fact that 8-Bromo-cGMP induced strong relaxation in the GPLP system. This is in contrast to the cGMP-dependent dilation shown by NCX 2057 in vascular system in a previous study where ODQ effectively prevented the induced vasodilation [15]. Nevertheless, nitrovasodilators have previously been shown to induce smooth muscle relaxation both dependent and independent via cGMP activation [40][41][42].
Although the tested substances are referred as NO donors, the effect of SNP and NCX 2057 in the peripheral lung may not only depend on NO generation. The concentrations of the pharmacological agents were higher than are ordinarily used in vivo and also higher than NO levels produced in vivo. Measurements of nitrite and nitrate indicated that SNP and NCX 2057 differed notably in their kinetic profile. NCX 2057 produced significantly more nitrite than SNP at early timepoints ( fig. 4). Nitrite accumulation is generally interpreted as an indicator of NO release. Thus, this could be a possible explanation for the decreased effectiveness of SNP relative to NCX 2057 as an inhibitor of lung contractility. However, it is important to realize that drugs may be metabolised to nitrate and nitrite without apparent formation of NO and therefore one cannot rely completely of nitrate/nitrite measurements as accurate estimates of NO formation. Several additional factors determine the rate of NO release and bioavailability of different NO donors including, lipophilicity, site of NO release, the presence of metal containing and thiol containing compounds, the stability of the NO bonds, and the propensity for homolytic vs. heterolytic cleavage [43]. In contrast to NCX 2057, SNP also transfers NO + [43,44], which can react with sulphydryl groups (-SH), to form S-nitrosothiols [45]. The mechanisms behind the distinct inhibitory effects of the tested NO donors on the early allergic airway response need to be further evaluated in other test systems.

Conclusion
The obtained results point at important differences between the tested NO donors, making it crucial to carefully characterize the profile of a selected NO donor, when using it as an experimental tool. The data revealed that the tested NO donors SNP and NCX 2057 acted as specific inhibitors of allergen-induced mediator release. In contrast to SNP, NCX 2057 also induced relaxation in the GPLP, but not via the cGMP pathway. The findings support that different NO donors may have specific antiinflammatory effects in the peripheral lung tissue and may facilitate the development of anti-inflammatory therapeutic strategies targeting distinct effects of early allergic airway response.
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