In rat alveolar macrophages lipopolysaccharides exert divergent effects on the transport of the cationic amino acids L-arginine and L-ornithine.
In rat alveolar macrophages (AMphi) 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 AMphi, could, via inhibition of L-arginine uptake, act as a paracrine inhibitor of NO synthesis. Rat AMphi (cultured for 20 h in the absence or presence of 1 microg/ml LPS) were incubated in Krebs-HEPES solution containing [3H]-L-arginine (0.1 microM for 2 min or 100 microM 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 microM or 100 microM) occurred independently of sodium ions and was inhibited by L-ornithine (EC50: 117 and 562 microM, respectively) and with similar potencies by L-lysine. In LPS-treated AMphi 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 microM) by 43 and 58%, respectively, and the effect of 0.1 mM L-leucine was partially sodium dependent. In LPS-treated AMphi, 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 microM (L-arginine) and 39 and 1050 microM (L-ornithine), respectively. After LPS treatment Km2 of L-arginine transport was reduced to 63 microM and Vmax of both components was increased, whereas Km2 of L-ornithine transport was enhanced to 1392 microM and Vmax1 reduced. LPS-stimulated AMphi, incubated in amino acid-free Krebs-HEPES solution, produced about 4 nmol nitrite/10(6) cells per 6 h, and L-arginine enhanced nitrite accumulation maximally about threefold (EC50: 30 microM). L-ornithine, up to 3 mM, failed to affect significantly nitrite accumulation observed in the presence of 30 or 100 microM L-arginine. Rat AMphi 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 AMphi 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.
Messeri Dreissig
M D
MD
Institut für Pharmakologie und Toxikologie der Universität Bonn, Germany.
Hammermann
R
R
Mössner
J
J
Göthert
M
M
Racké
K
K
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D001992:Bronchoalveolar Lavage Fluid; D002478:Cells, Cultured; D007930:Leucine / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008239:Lysine / Q000378:metabolism*; D016676:Macrophages, Alveolar / Q000201:enzymology / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009952:Ornithine / Q000378:metabolism*; D012333:RNA, Messenger / Q000302:isolation & purification; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D001120:Arginine; D012964:Sodium; D009952:Ornithine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D007930:Leucine; D008239:Lysine