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

Abstract

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.

Arginine Transport via Cationic Amino Acid Transporter 2 Plays a Critical Regulatory Role in Classical or Alternative Activation of Macrophages

“…In peritoneal macrophages, this transport is induced by LPS ϩ IFN-␥ through the y ϩ system and particularly by CAT2 (14), while in human monocytes, IFN-␥ stimulates this transport through the y ϩ L system (33). In rat alveolar macrophages, the y ϩ and y ϩ L transport systems participate in arginine transport (28). Previous observations in rat alveolar macrophages described an increase in CAT2 after LPS treatment (28).…”

“…In rat alveolar macrophages, the y ϩ and y ϩ L transport systems participate in arginine transport (28). Previous observations in rat alveolar macrophages described an increase in CAT2 after LPS treatment (28).…”

Arginine and Asthma

“…In particular, upregulation of CAT-2B has been associated with increased L-arginine uptake under conditions of iNOS induction stimulated by proinflammatory mediators [46][47][48]. This suggests that, at least in some cells, increased uptake may offset reductions in circulating arginine levels.…”

Arginase in Asthma – Recent Developments in Animal and Human Studies~!2009-11-12~!2010-03-23~!2010-05-04~!

“…The bioavailability of Larginine to NOS could also be regulated by the arginase product L-ornithine, which competitively inhibits the uptake of L-arginine via specific CATs of the y + -system [46][47][48]. Consistent with this finding, incubation with L-ornithine increased airway responsiveness in perfused guinea pig tracheal preparations by inducing a deficiency of cNOSderived NO [49].…”

“…In conclusion, increased arginase activity underlies the deficiency of neuronal and nonneuronal NO and subsequent AHR after the EAR by limiting the substrate availability to cNOS isoforms. Of note, since L-arginine transport via the y + -system is inhibited by L-ornithine [46][47][48] leading to a deficiency of NO and increased airway responsiveness [49], increased formation of L-ornithine by arginase could also contribute to the L-arginine limitation and NO deficiency in allergic asthma.…”