Arginase strongly impairs neuronal nitric oxide-mediated airway smooth muscle relaxation in allergic asthma

Abstract

Background: Using guinea pig tracheal preparations, we have recently shown that endogenous arginase activity attenuates inhibitory nonadrenergic noncholinergic (iNANC) nerve-mediated airway smooth muscle relaxation by reducing nitric oxide (NO) production--due to competition with neuronal NO-synthase (nNOS) for the common substrate, L-arginine. Furthermore, in a guinea pig model of allergic asthma, airway arginase activity is markedly increased after the early asthmatic reaction (EAR), leading to deficiency of agonist-induced, epithelium-derived NO and subsequent airway hyperreactivity. In this study, we investigated whether increased arginase activity after the EAR affects iNANC nerve-derived NO production and airway smooth muscle relaxation.

Methods: Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5-16 Hz)-induced relaxation was measured in tracheal open-ring preparations precontracted to 30% with histamine in the presence of 1 microM atropine and 3 microM indomethacin. The contribution of NO to EFS-induced relaxation was assessed by the nonselective NOS inhibitor Nomega-nitro-L-arginine (L-NNA, 100 microM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor Nomega-hydroxy-nor-L-arginine (nor-NOHA, 10 microM). Furthermore, the role of substrate availability to nNOS was measured in the presence of exogenous L-arginine (5.0 mM).

Results: At 6 h after ovalbumin-challenge (after the EAR), EFS-induced relaxation (ranging from 3.2 +/- 1.1% at 0.5 Hz to 58.5 +/- 2.2% at 16 Hz) was significantly decreased compared to unchallenged controls (7.1 +/- 0.8% to 75.8 +/- 0.7%; P < 0.05 all). In contrast to unchallenged controls, the NOS inhibitor L-NNA did not affect EFS-induced relaxation after allergen challenge, indicating that NO deficiency underlies the impaired relaxation. Remarkably, the specific arginase inhibitor nor-NOHA normalized the impaired relaxation to unchallenged control (P < 0.05 all), which effect was inhibited by L-NNA (P < 0.01 all). Moreover, the effect of nor-NOHA was mimicked by exogenous L-arginine.

Conclusion: The results clearly demonstrate that increased arginase activity after the allergen-induced EAR contributes to a deficiency of iNANC nerve-derived NO and decreased airway smooth muscle relaxation, presumably via increased substrate competition with nNOS.

Figure 1

iNANC nerve-induced airway smooth muscle relaxation is impaired after allergen challenge due to deficiency of nNOS-derived NO. Electrical field stimulation-induced relaxation of precontracted tracheal open-ring preparations obtained from unchallenged and from OA-challenged guinea pigs in the absence and presence of the NOS inhibitor L-NNA (100 μM). Results are means ± s.e.m. of 7 experiments for each condition. *P < 0.05 and **P < 0.01 compared to unchallenged control, n.s. = non significant.

Figure 2

Role of arginase and NO in impaired iNANC nerve-induced relaxation of guinea pig tracheal smooth muscle after allergen-challenge. Electrical field stimulation-induced relaxation of precontracted tracheal open-ring preparations obtained from unchallenged and from OA-challenged guinea pigs in the absence and presence of the arginase inhibitor nor-NOHA (10 μM), with or without the NOS inhibitor L-NNA (100 μM). Results are means ± s.e.m. of 7 experiments for each condition. *P < 0.05 and **P < 0.01 compared to unchallenged control, ^†P < 0.05 and ^‡P < 0.01 compared to OA-challenged control, ^#P < 0.01 compared to nor-NOHA-treated.

Figure 3

Role of L-arginine availability in impaired iNANC nerve-induced relaxation of guinea pig tracheal smooth muscle after allergen-challenge. Electrical field stimulation-induced relaxation of precontracted tracheal open-ring preparations obtained from unchallenged and from OA-challenged guinea pigs in the absence and presence of L-arginine (5.0 mM). Results are means ± s.e.m. of 4 (OA-challenged) to 7 (unchallenged) experiments. *P < 0.05 and **P < 0.01 compared to unchallenged control, ^†P < 0.05 compared to OA-challenged control.