Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages

Abstract

We assessed whether reactive oxygen-nitrogen intermediates generated by alveolar macrophages (AMs) oxidized and nitrated human surfactant protein (SP) A. SP-A was exposed to lipopolysaccharide (100 ng/ml)-activated AMs in 15 mM HEPES (pH 7.4) for 30 min in the presence and absence of 1.2 mM CO2. In the presence of CO2, lipopolysaccharide-stimulated AMs had significantly higher nitric oxide synthase (NOS) activity (as quantified by the conversion ofl-[U-14C]arginine tol-[U-14C]citrulline) and secreted threefold higher levels of nitrate plus nitrite in the medium [28 ± 3 vs. 6 ± 1 (SE) nmol ⋅ 6.5 h−1 ⋅ 106AMs−1]. Western blotting studies of immunoprecipitated SP-A indicated that CO2 enhanced SP-A nitration by AMs and decreased carbonyl formation. CO2(0–1.2 mM) also augmented peroxynitrite (0.5 mM)-induced SP-A nitration in a dose-dependent fashion. Peroxynitrite decreased the ability of SP-A to aggregate lipids, and this inhibition was augmented by 1.2 mM CO2. Mass spectrometry analysis of chymotryptic fragments of peroxynitrite-exposed SP-A showed nitration of two tyrosines (Tyr164 and Tyr166) in the absence of CO2 and three tyrosines (Tyr164, Tyr166, and Tyr161) in the presence of 1.2 mM CO2. These findings indicate that physiological levels of peroxynitrite, produced by activated AMs, nitrate SP-A and that CO2 increased nitration, at least partially, by enhancing enzymatic nitric oxide production.