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

We assessed whether reactive oxygen-nitrogen intermediates generated by alv eolar 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 hi gher nitric oxide synthase (NOS) activity (as quantified by the conversion of L-[U-C-14]arginine to L-[U-C-14]citrulline) and secreted threefold highe r levels of nitrate plus nitrite in the medium [28 +/- 3 vs. 6 +/- 1 (SE) n mol 6.5 h(-1) 10(6) AMs(-1)]. Western blotting studies of immunoprecipitate d SP-A indicated that CO2 enhanced SP-A nitration by AMs and decreased carb onyl formation. CO2 (0-1.2 mM) also augmented peroxynitrite (0.5 mM)induced SP-A nitration in a dose-dependent fashion. Peroxynitrite decreased the ab ility of SP-A to aggregate lipids, and this inhibition was augmented by 1.2 mM CO2. Mass spectrometry analysis of chymotryptic fragments of peroxynitr ite-exposed SP-A showed nitration of two tyrosines (Tyr(164) and Tyr(166)) in the absence of CO2 and three tyrosines (Tyr164 Tyr(166), and Tyr(161)) i n the presence of 1.2 mM CO2. These findings indicate that physiological le vels of peroxynitrite, produced by activated AMs, nitrate SP-A and that CO2 increased nitration, at least partially, by enhancing enzymatic nitric oxi de production.