The hypothesis was investigated that the nitric oxide (NO) synthase intermediate, N%hydroxy-L-arginine (HOArg), is an arginase inhibitor in rabbit or rat alveolar macrophages. Exogenously applied HOArg strongly inhibited the arginase activity present in these cells (ICs0 > 15 ItM), and attenuated L-[3Hlarginine transport (ICs0 -> 500 ~M) in rabbit alveolar macrophages. Moreover, up to 37 ~M HOArg were detected in the conditioned medium, but not in the lysate, of rat alveolar macrophages exposed to bacterial lipopolysaccharide for 18 h. HOArg may thus be a potent endogenous arginase inhibitor in these cells which increases the availability of L-arginine for NO biosynthesis.

1 Alveolar macrophages (AMF) exhibit arginase activity and may, in addition, express an inducible form of nitric oxide (NO) synthase (iNOS). Both pathways may compete for the substrate, L-arginine. The present study tested whether two recently described potent inhibitors of liver arginase (N o -hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine) might also inhibit arginase in AMF and whether inhibition of arginase might a ect L-arginine utilization by iNOS. 2 AMF obtained by broncho-alveolar lavage of rat and rabbit isolated lungs were disseminated (2.5 or 3610 6 cells per well) and allowed to adhere for 2 h. Thereafter, they were either used to study In conclusion, N o -hydroxy-D,L-indospicine is a potent and speci®c inhibitor of arginase in AMF. In cells in which, in addition to arginase, iNOS is expressed, inhibition of arginase can cause a shift of Larginine metabolism to the NOS pathway. However, the extent of this shift appears to depend in a complex manner on the level of iNOS.

Alveolar macrophages were obtained by broncho-alveolar lavage of isolated rat and rabbit lungs and cultured (2.5 x 10(6) cells/dish) for 18 h in the absence or presence of bacterial lipopolysaccharides (LPS) alone or in combination with cytokines. Thereafter, accumulation of 3H-citrulline (NO synthase activity) and 3H-ornithine (arginase activity) were determined. During incubation of rat alveolar macrophages with 3H-arginine clear amounts of 3H-citrulline and 3H-ornithine (3.8 and 4.6% of the added 3H-arginine, respectively) were formed and most of these metabolites appeared in the incubation medium (ratios extra-/intracellular of 17 and 70 for 3H-citrulline and 3H-ornithine, respectively). When rat alveolar macrophages had been cultured with LPS the formation of 3H-citrulline was increased about 30-fold and this was accompanied by a reduction in 3H-ornithine formation of about 60%. The effects of LPS were largely attenuated by dexamethasone (10 mumol/l). Inhibition of NO synthase by NG-monomethyl-L-arginine (L-NMMA, 100 mumol/l) in LPS treated alveolar macrophages reduced the formation 3H-citrulline by more than 90% and restored the 3H-ornithine formation. After culturing in the presence of LPS the ratios extra/intracellular of 3H-citrulline and 3H-ornithine were markedly enhanced and this effect was not dexamethasone sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)

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 (AMPhi) and tracheal epithelial cells, and whether L-arginine-dependent pathways were affected. The effect of cationic polypeptides on cellular uptake of [(3)H]-L-arginine, nitrite accumulation, and the turnover of [(3)H]-L-arginine by nitric oxide (NO) synthase and arginase (formation of [(3)H]-L-citrulline and [(3)H]-L-ornithine, respectively) were studied. Poly-L-arginine reduced [(3)H]-L-arginine uptake in rat AMPhi and tracheal epithelial cells in a concentration-dependent manner (at 300 microgram/ml by 70%). Poly-L-lysine, protamine, and major basic protein (each up to 300 microgram/ml) tested in rat AMPhi inhibited [(3)H]-L-arginine uptake by 35 to 50%. During 6 h incubation in amino acid-free Krebs solution, rat AMPhi, precultured in the absence or presence of LPS (1 microgram/ml), accumulated 1.4 and 3.5 nmol/10(6) cells nitrite, respectively. Addition of 100 microM L-arginine increased nitrite accumulation by 70 and 400% in control and lipopolysaccharide-treated AMPhi, respectively. Nitrite accumulation in the presence of L-arginine was reduced by poly-L-arginine and poly-L-lysine (100 and 300 microgram/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 microgram/ml) inhibited [(3)H]-L-citrulline formation by AMPhi stronger than that of [(3)H]-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.

6. ¬_Leucine (0·1 mÒ) inhibited ¬_[ 3 H]arginine uptake by 50% in sodium-containing medium, but not in sodium-free medium. At 1 mÒ, ¬_leucine caused significant inhibition in sodiumfree medium also. ¬_Leucine showed similar effects on PMA-treated cells. 7. N-Ethylmaleimide (200 ìÒ, 10 min) reduced ¬_[ 3 H]arginine uptake by 70% in control cells, but had no effect on PMA-treated (20 or 2 h) cells. 8. In alveolar macrophages, multiple transport systems are involved in ¬_arginine uptake, which is markedly stimulated by protein kinase C, probably by modulation of the activity of already expressed cationic amino acid transporters.7946