Am. J. Respir. Cell Mol. Biol., Volume 21, Number 2, August, 1999 155-162

Cationic Proteins Inhibit L-Arginine Uptake in Rat Alveolar Macrophages and Tracheal Epithelial Cells
Implications for Nitric Oxide Synthesis

Rainer Hammermann, Joachim Hirschmann, Claudia Hey, Jutta Mössner, Gert Folkerts, Frans P. Nijkamp, Ignaz Wessler, and Kurt Racké

Institute of Pharmacology and Toxicology, University of Bonn, Bonn; Department of Pharmacology, University of Mainz, Mainz, Germany; and Faculty of Pharmacy, University of Utrecht, Utrecht, The Netherlands

Eosinophil-derived cationic proteins play an essential role in the pathogenesis of bronchial asthma. We tested whether cationic proteins interfere with the cationic amino-acid transport in alveolar macrophages (AM) and tracheal epithelial cells, and whether L-arginine-dependent pathways were affected. The effect of cationic polypeptides on cellular uptake of [³H]-L-arginine, nitrite accumulation, and the turnover of [³H]-L-arginine by nitric oxide (NO) synthase and arginase (formation of [³H]-L-citrulline and [³H]-L-ornithine, respectively) were studied. Poly-L-arginine reduced [³H]-L-arginine uptake in rat AM and tracheal epithelial cells in a concentration-dependent manner (at 300 µg/ml by 70%). Poly-L-lysine, protamine, and major basic protein (each up to 300 µg/ml) tested in rat AM inhibited [³H]-L-arginine uptake by 35 to 50%. During 6 h incubation in amino acid-free Krebs solution, rat AM, precultured in the absence or presence of LPS (1 µg/ml), accumulated 1.4 and 3.5 nmol/10⁶ cells nitrite, respectively. Addition of 100 µM L-arginine increased nitrite accumulation by 70 and 400% in control and lipopolysaccharide-treated AM, respectively. Nitrite accumulation in the presence of L-arginine was reduced by poly-L-arginine and poly-L-lysine (100 and 300 µg/ml) by 60 to 85% and 20 to 30%, respectively. Poly-L-arginine, but not poly-L-lysine, inhibited nitrite accumulation already in the absence of extracellular L-arginine. Poly-L-arginine (300 µg/ml) inhibited [³H]-L-citrulline formation by AM stronger than that of [³H]-L-ornithine. We conclude that cationic proteins can inhibit cellular transport of L-arginine and this can limit NO synthesis. Poly-L-arginine inhibits L-arginine uptake more effectively than other cationic proteins and exerts additional direct inhibitory effects on NO synthesis.

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