In rat alveolar macrophages lipopolysaccharides exert divergent effects on the transport of the cationic amino acids l-arginine and l-ornithine
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In rat alveolar macrophages (AMΦ) it was tested whether induction of iNOS by lipopolysaccharides (LPS) is accompanied by changes in l-arginine transport and whether l-ornithine, the product of arginase released from AMΦ, could, via inhibition of l-arginine uptake, act as a paracrine inhibitor of NO synthesis.
Rat AMΦ (cultured for 20 h in the absence or presence of 1 µg/ml LPS) were incubated in Krebs-HEPES solution containing [3H]-l-arginine (0.1 µM for 2 min or 100 µM for 5 min) and the cellular radioactivity was determined as a measure of l-arginine uptake. In parallel, cells were incubated for 6 h in Krebs-HEPES solution containing 0–1 mM l-arginine and nitrite accumulation was determined. [3H]-l-Arginine uptake (0.1 µM or 100 µM) occurred independently of sodium ions and was inhibited by l-ornithine (EC50: 117 and 562 µM, respectively) and with similar potencies by l-lysine. In LPS-treated AMΦ the concentration inhibition curve of l-ornithine was shifted to the right by about a factor of 4, whereas that of l-lysine was only marginally shifted to the right. l-Leucine (0.1 and 1 mM) inhibited [3H]-l-arginine (0.1 µM) by 43 and 58%, respectively, and the effect of 0.1 mM l-leucine was partially sodium dependent. In LPS-treated AMΦ, 0.1 mM l-leucine no longer inhibited [3H]-l-arginine and the effect of 1 mM l-leucine was attenuated. Kinetic analysis of the transport of [3H]-l-arginine and [14C]-l-ornithine revealed two components for each amino acid with Km values of 21 and 114 µM (l-arginine) and 39 and 1050 µM (l-ornithine), respectively. After LPS treatment Km2 of l-arginine transport was reduced to 63 µM and Vmax of both components was increased, whereas Km2 of l-ornithine transport was enhanced to 1392 µM and Vmax1 reduced. LPS-stimulated AMΦ, incubated in amino acid-free Krebs-HEPES solution, produced about 4 nmol nitrite/106 cells per 6 h, and l-arginine enhanced nitrite accumulation maximally about threefold (EC50: 30 µM). l-Ornithine, up to 3 mM, failed to affect significantly nitrite accumulation observed in the presence of 30 or 100 µM l-arginine. Rat AMΦ express mRNA for two cationic amino acid transporters (CAT-1 and CAT-2B), and LPS markedly up-regulated mRNA for CAT-2B in parallel with mRNA for iNOS, but had no effect on that for CAT-1.
In conclusion, in rat AMΦ LPS up-regulates l-arginine transport and induces changes in the characteristics of the cationic amino acid transport resulting in preferential transport of l-arginine. These effects may be regarded as cellular measures to ensure a high l-arginine supply for iNOS.