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Am J Physiol Lung Cell Mol Physiol 278: L1025-L1031, 2000;
1040-0605/00 $5.00
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Vol. 278, Issue 5, L1025-L1031, May 2000

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

Sha Zhu1,*, Khaled F. Basiouny1,*, John P. Crow1,2, and Sadis Matalon1,3,4

Departments of 1 Anesthesiology, 2 Pharmacology and Toxicology, 3 Physiology and Biophysics, and 4 Comparative Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35233-6810

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 of L-[U-14C]arginine to L-[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 · 106 AMs-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.

nitric oxide; peroxynitrite; nitrotyrosine; protein oxidation; alveolar epithelium; acute respiratory distress syndrome; hypercapnia; mass spectrometry

* S. Zhu and K. F. Basiouny contributed equally to this work.
  The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.




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