Heparin normalizes allergen-induced nitric oxide deficiency and airway hyperresponsiveness

Abstract

It has been established that polycations cause airway hyperresponsiveness (AHR) to methacholine by inducing a deficiency of constitutive nitric oxide synthase (cNOS)-derived bronchodilating nitric oxide (NO). Since a deficiency of cNOS-derived NO also contributes to allergen-induced AHR after the early asthmatic reaction (EAR) and since this AHR is associated with the release of polycationic proteins from infiltrated eosinophils in the airways, we hypothesized that endogenous polycations underlie or at least contribute to the allergen-induced NO deficiency and AHR. Using a guinea-pig model of allergic asthma, we addressed this hypothesis by examining the effect of the polyanion heparin, acting as a polycation antagonist, on the responsiveness to methacholine of isolated perfused tracheae from unchallenged control animals and from animals 6 h after ovalbumin challenge, that is, after the EAR. A 2.0-fold AHR (P<0.001) to intraluminal administration of methacholine was observed in airways from allergen-challenged animals compared to control. Incubation of these airways with 250 U ml(-1) heparin completely normalized the observed hyperresponsiveness (P<0.001), whereas the responsiveness to methacholine of airways from unchallenged control animals was not affected. The effect of heparin on airways from allergen-challenged guinea-pigs was dose-dependently (0.1 and 1.0 mM) reversed by the NOS inhibitor L-NAME (P<0.01). These results indicate that endogenous (presumably eosinophil-derived) polycations are involved in allergen-induced NO deficiency and AHR after the EAR, probably by inhibition of l-arginine transport.

Figure 1

Methacholine (MeCh; IL)-induced constriction of intact perfused tracheal preparations obtained from (a) unchallenged and (b) ovalbumin (OA)-challenged guinea-pigs, in the absence and presence of 250 U ml⁻¹ heparin. Results are the means±s.e.m. of 5–8 experiments.

Figure 2

Methacholine (MeCh; IL)-induced constriction of intact perfused tracheae from ovalbumin (OA)-challenged guinea-pigs, in the absence and presence of 250 U ml⁻¹ heparin, alone or in combination with 0.1 or 1.0 mM L-NAME. Results are the means±s.e.m. of 4–8 experiments.

Figure 3

Spontaneous EPO release from BAL eosinophils obtained from sensitized guinea-pigs at 6 h after saline or ovalbumin (OA) challenge, expressed as the % of total cellular EPO content. 100% represents 21.9±5.2 and 22.5±3.6 ng ml⁻¹ EPO 10⁶ cells⁻¹ for saline- and OA-challenged animals, respectively. Results are the means±s.e.m. of 5–8 experiments. ^*P<0.001 compared to saline.