Localization and distribution of NOS1 in murine airways

Nitric oxide synthase 1 (NOS1) is a major determinant of bronchial responsiveness in mice and has been proposed as an asthma gene in man. Nevertheless, how nitric oxide production by NOS1 contributes to airway responsiveness remains unclear. Although NOS1 is usually closely associated with nerves, it has also been found in a variety of other cell types, particularly epithelium. We sought to better understand the role of NOS1 by determining its major site of expression in murine airways. Using nicotinamide adenine dinucleotide phosphate-diaphorase (diaphorase), which non-selectively detects nitric oxide synthase (NOS), we found strong evidence of NOS in the airways largely restricted to the airway epithelium and trachea glands. In contrast, diaphorase staining of NOS1-deficient mutant mice demonstrated a marked reduction in epithelial cells of the trachea but not bronchioles, suggesting that the epithelium is the major site of NOS1 expression. This was supported by immunohistochemistry, which also demonstrated significant staining in glands and to a lesser degree in airway smooth muscle. Double immunofluorescence staining of tracheas for NOS1 and the nerve marker PGP 9.5 failed to demonstrate co-localization, indicating that nerves are not an important source of NOS1 in the murine airway wall. Finally, removal of the trachea epithelium by digestion resulted in a marked decrease in NOS1 detection by Western blotting, confirming the epithelium as the major site of NOS1 expression in the murine airway. These findings support the notion that the role of NOS1 in murine bronchial responsiveness involves the epithelium of the central airways.