Increased arginase activity underlies allergen-induced deficiency of cNOS-derived nitric oxide and airway hyperresponsiveness

Abstract

1. A deficiency of constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO), due to reduced availability of L-arginine, importantly contributes to allergen-induced airway hyperresponsiveness (AHR) after the early asthmatic reaction (EAR). Since cNOS and arginase use L-arginine as a common substrate, we hypothesized that increased arginase activity is involved in the allergen-induced NO deficiency and AHR. 2. Using a guinea-pig model of allergic asthma, we addressed this hypothesis by examining the effects of the specific arginase inhibitor N(omega)-hydroxy-nor-L-arginine (nor-NOHA) on the responsiveness to methacholine of isolated perfused tracheae from unchallenged control animals and from animals 6 h after ovalbumin challenge. Arginase activity in these preparations was investigated by measuring the conversion of L-[14C]arginine to [14C]urea. 3. Airways from allergen-challenged animals showed a 2 fold (P<0.001) increase in responsiveness to intraluminal (IL) administration of methacholine compared to controls. A similar hyperresponsiveness (1.8 fold, P<0.01) was observed in control airways incubated with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 0.1 mM, IL), while L-NAME had no further effect on the airways from challenged animals. 4. Remarkably, 5 microM nor-NOHA (IL) normalized the hyperresponsiveness of challenged airways to basal control (P<0.001), and this effect was fully reversed again by 0.1 mM L-NAME (P<0.05). Moreover, arginase activity in homogenates of the hyperresponsive airways was 3.5 fold (P<0.001) enhanced compared to controls. 5. The results indicate that enhanced arginase activity contributes to allergen-induced deficiency of cNOS-derived NO and AHR after the EAR, presumably by competition with cNOS for the common substrate, L-arginine. This is the first demonstration that arginase is involved in the pathophysiology of asthma.

Figure 1

Methacholine (MeCh; IL)-induced constriction of intact perfused tracheal preparations obtained from (a) unchallenged guinea-pigs, in the absence (control) and presence of 0.1 mM L-NAME, 5.0 μM nor-NOHA, and 5.0 μM nor-NOHA plus 0.1, 0.5 or 1.0 mM L-NAME, and (b) ovalbumin (OA)-challenged guinea-pigs, in the absence (control) and presence of 5.0 and 10.0 μM nor-NOHA, 0.1 mM L-NAME, and 5.0 μM nor-NOHA plus 0.1 mM L-NAME. For comparison, methacholine-induced constriction of control preparations from unchallenged guinea-pigs is also shown in b. Methacholine, nor-NOHA and L-NAME were all applied to the IL compartment. Results are means±s.e.mean of 3 – 14 experiments.

Figure 2

Arginase activity in tracheal homogenates from unchallenged and ovalbumin (OA)-challenged guinea-pigs, in the absence (control) and presence of 0.1 mM L-NAME and 5.0 μM nor-NOHA. Results are means±s.e.mean of 3 – 7 experiments. ^*P<0.001, †P<0.0001 compared to unchallenged control; ‡P<0.005 compared to OA-challenged control.