In rat alveolar macrophages lipopolysaccharides exert divergent effects onthe transport of the cationic amino acids L-arginine and L-ornithine

In rat alveolar macrophages (AM Phi) it was tested whether induction of iNO S by lipopolysaccharides (LPS) is accompanied by changes in L-arginine tran sport and whether L-ornithine, the product of arginase released from AM Phi , could, via inhibition of L-arginine uptake. act as a paracrine inhibitor of NO synthesis. Rat AM Phi (cultured for 20 h in the absence or presence of 1 mu g/ml LPS) were incubated in Krebs-HEPES solution containing [H-3]-L-arginine (0.1 mu M for 2 min or 100 mu M for 5 min) ansi the cellular radioactivity was dete rmined 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 a ccumulation was determined. [H-3]-L-Arginine uptake (0.1 mu M or 100 mu M) occur-red independently of sodium ions and was inhibited by L-ornithine (EC 50: 117 and 562 mu M, respectively) and with similar potencies by L-lysine. In LPS-treated AM Phi the concentration inhibition curve of L-ornithine wa s 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 [H-3]-L-arginine (0.1 CIM) by 43 and 58%, respectively, and the effect of 0 .1 mM L-leucine was partially sodium dependent. In LPS-treated AM Phi, 0.1 mM L-leucine no longer inhibited [H-3]-L-arginine and the effect of 1 mM L- leucine was attenuated. Kinetic analysis of the transport of [3H]-L-arginin e and [C-14]-L-ornithine revealed two components for each amino acid with K -m values of 21 and 114 mu M (L-arginine) and 39 and 1050 mu M (L-ornithine ), respectively. After LPS treatment K-m2 of L-arginine transport was reduc ed to 63 mu M and V-max of both components was increased, whereas K-m2 of L -ornithine transport was enhanced to 1392 mu M and V-max reduced. LPS-stimu lated AM Phi, incubated in amino acid-free Krebs-HEPES solution, produced a bout 4 nmol nitrite/10(6) cells per 6 h, and L-arginine enhanced nitrite ac cumulation maximally about threefold (EC50: 30 mu M). L-Ornithine, up to 3 mM, failed to affect significantly nitrite accumulation observed in the pr esence of 30 or 100 mu M L-arginine. Rat AM Phi express mRNA for two cation ic amino acid transporters (CAT-1 and CAT-2B), and LPS markedly up-regulate d mRNA for CAT-2B in parallel with mRNA for iNOS, but had no effect on that for CAT-1. In conclusion, in rat AM Phi LPS up-regulates L-arginine transport and indu ces changes in the characteristics of the cationic amino acid transport res ulting in preferential transport of L-arginine. These effects may be regard ed as cellular measures to ensure a high L-arginine supply for iNOS.