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

Abstract

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 microM, p < 0.001) and SNP (100 microM, p < 0.05). Antigen-induced eicosanoid release was decreased by NCX 2057 (100 microM, p < 0.001) but not by SNP (100 microM), whereas the release of histamine was reduced by SNP (100 microM, p < 0.001) but not by NCX 2057 (100 microM). In addition, NCX 2057 (0.1-100 microM), but not SNP (0.1-100 microM), 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.

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.

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.

Figure 3

Effect of pretreatment with different drugs compared with the control on mediator release from sensitized GPLP after challenge with 1000 ng/ml of OVA. Release of A, Histamine (ng/ml); B, CysLTs; C, LTB₄; D, PGD₂; E, TXB₂ (pg/ml). All samples were collected at baseline and then at the plateau after 1000 ng/ml of OVA. The parenchymal strips had been pretreated for 15 min with Tyrode's solution (control), 100 μM SNP (SNP), 100 μM NCX 2057 (2057) and 100 μM ferulic acid (FA). All data are expressed as mean ± s.e.m.; statistical analysis was performed by one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs control.

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.

Figure 5

A, Generation of nitrite (nM) over two hours in the GPLP organ bath fluid after addition of SNP (100 μM; n = 3), NCX 2057 (100 μM; n = 3), DMSO (n = 3) or Tyrode's buffer (n = 3). B, Generation of nitrate over two hours in the GPLP organ bath fluid after addition of NCX 2057 (100 μM; n = 3), DMSO (n = 3) or Tyrode's buffer (n = 3). Data are expressed as mean ± s.e.m.

Figure 6

Effect of cumulative concentrations of 8-bromo-cGMP (0.1–100 μM, n = 3), SNP (0.1–100 μM, n = 4), NCX 2057 (0.1–100 μM) alone (n = 6) and in combination with 30 μM ODQ (n = 3) compared to control (n = 5) on contractions induced by 10 nM of LTD₄ on GPLP. Data are expressed as mean ± s.e.m.; statistical analysis was performed by two-way ANOVA. *P < 0.05;^##, **P < 0.01; ***P < 0.001 vs control.

Figure 7

Effect of pretreatment with NCX 2057 (1 μM, n = 4; 10 μM, n = 5; 100 μM, n = 5) on the concentration-response to LTD₄ (0.1–100 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.