Contribution of nitric oxide synthases 1, 2, and 3 to airway hyperresponsiveness and inflammation in a murine model of asthma

Abstract

Asthma is a chronic disease characterized by increased airway responsiveness and airway inflammation. The functional role of nitric oxide (NO) and the various nitric oxide synthase (NOS) isoforms in human asthma is controversial. To investigate the role of NO in an established model of allergic asthma, mice with targeted deletions of the three known isoforms of NOS (NOS1, 2, and 3) were studied. Although the inducible (NOS2) isoform was significantly upregulated in the lungs of ovalbumin (OVA)-sensitized and -challenged (OVA/OVA) wild-type (WT) mice and was undetectable in similarly treated NOS2-deficient mice, airway responsiveness was not significantly different between these groups. OVA/OVA endothelial (NOS3)-deficient mice were significantly more responsive to methacholine challenge compared with similarly treated NOS1 and NOS1&3-deficient mice. Airway responsiveness in OVA/OVA neuronal (NOS1)-deficient and neuronal/endothelial (NOS1&3) double-deficient mice was significantly less than that observed in similarly treated NOS2 and WT groups. These findings demonstrate an important function for the nNOS isoform in controlling the inducibility of airway hyperresponsiveness in this model of allergic asthma.

Figure 1

Assessment of calcium-dependent (cNOS, eNOS, and nNOS activity) and calcium-independent (iNOS activity) pulmonary NOS activity in OVA/PBS and OVA/OVA WT and NOS-deficient mice. Calcium-dependent (top) and -independent (bottom) NOS activity was measured in whole lung preparations as described in Materials and Methods. Data represents means ± SEM. ^# P < 0.05 compared with OVA/PBS, same genotype. ^¶ P < 0.05 compared with WT, same treatment. Black bars, OVA/OVA; hatched bars, OVA/PBS.

Figure 2

(A) Airway responsiveness measured as ED₂₀₀R_L in anesthetized OVA/OVA WT (SV129/B6) and NOS1-, NOS3-, and NOS1&3-deficient mice (bred on a SV129/B6 background). Airway responses were measured from the methacholine dose–response curves. The dose necessary to cause a doubling of lung resistance was calculated by log linear interpolation. (B) Airway responses measured in anesthetized OVA/OVA WT (B6) and OVA/OVA NOS2-deficient mice bred on a C57BL/6 (B6) background. The log ED₂₀₀R_L values represent an index of airway responsiveness. There were no significant differences between the OVA/OVA WT (B6) and OVA/ OVA NOS2-deficient mice. Numerically lower values are indicative of increased airway responsiveness. Data represents mean log ED₂₀₀R_L values ± SEM.

Figure 2

(A) Airway responsiveness measured as ED₂₀₀R_L in anesthetized OVA/OVA WT (SV129/B6) and NOS1-, NOS3-, and NOS1&3-deficient mice (bred on a SV129/B6 background). Airway responses were measured from the methacholine dose–response curves. The dose necessary to cause a doubling of lung resistance was calculated by log linear interpolation. (B) Airway responses measured in anesthetized OVA/OVA WT (B6) and OVA/OVA NOS2-deficient mice bred on a C57BL/6 (B6) background. The log ED₂₀₀R_L values represent an index of airway responsiveness. There were no significant differences between the OVA/OVA WT (B6) and OVA/ OVA NOS2-deficient mice. Numerically lower values are indicative of increased airway responsiveness. Data represents mean log ED₂₀₀R_L values ± SEM.

Figure 3

Airway responsiveness measured by whole body plethysmography in awake WT (B6) and NOS2-KO mice. Penh, an index of airway obstruction, was calculated from the box pressure/time wave form after aerosolization of increasing doses of methacholine. Numerically higher values of Penh are indicative of increased airway obstruction. Dose– response curves are shown for OVA- and PBS-challenged WT and NOS2-KO mice. Data represents mean Penh values ± SEM.