Inhibition of arginase in rat and rabbit alveolar macrophages by N omega-hydroxy-D,L-indospicine, effects on L-arginine utilization by nitric oxide synthase.
Résumé
1. Alveolar macrophages (AM phi) 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 omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine) might also inhibit arginase in AM phi and whether inhibition of arginase might affect L-arginine utilization by iNOS. 2. AM phi obtained by broncho-alveolar lavage of rat and rabbit isolated lungs were disseminated (2.5 or 3 x 10(6) cells per well) and allowed to adhere for 2 h. Thereafter, they were either used to study [3H]-L-arginine uptake (37 kBq, 0.1 microM, 2 min) or cultured for 20 h in the absence or presence of bacterial lipopolysaccharide (LPS). Cultured AM phi were incubated for 1 h with [3H]-L-arginine (37 kBq, 0.1 microM) and the accumulation of [3H]-L-citrulline (NOS activity) and [3H]-L-ornithine (arginase activity) was determined. 3. During 1 h incubation of rabbit AM phi with [3H]-L-arginine, no [3H]-L-citrulline, but significant amounts of [3H]-L-ornithine (150 d.p.m x 1000) were formed. N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine, present during incubation, concentration-dependently reduced [3H]-L-ornithine formation (IC50: 2 and 45 microM, respectively). 4. N omega-hydroxy-D,L-indospicine (up to 100 microM) had no effect on [3H]-L-arginine uptake into rabbit AM phi, whereas 4-hydroxyamidino-D,L-phenylalanine caused a concentration-dependent inhibition (IC50: 300 microM). 5. Rat AM phi, cultured in the absence of LPS, formed significant amounts of [3H]-L-citrulline and [3H]-L-ornithine (133 and 212 d.p.m x 1000, respectively) when incubated for 1 h with [3H]-L-arginine. When AM phi had been cultured in the presence of 0.1 or 1 microgram ml-1 LPS, the formation of [3H]-L-citrulline was enhanced by 37 +/- 8.3 and 99 +/- 12% and that of [3H]-L-ornithine reduced by 21 +/- 8.7 and 70 +/- 2.5%, respectively. 6. In rat AM phi, cultured in the absence or presence of LPS, N omega-hydroxy-D,L-indospicine (10 and 30 microM) greatly reduced formation of [3H]-L-ornithine (by 80-95%) and this was accompanied by increased formation of [3H]-L-citrulline. However, only 20-30% of the [3H]-L-arginine not metabolized to [3H]-L-ornithine after inhibition of arginase was metabolized to [3H]-L-citrulline, when the AM phi had been cultured in the absence of LPS (i.e. low level of iNOS). On the other hand, when the AM phi had been cultured in the presence of LPS (i.e. high level of iNOS), all the [3H]-L-arginine not metabolized by the inhibited arginase was metabolized to [3H]-L-citrulline. 7. In conclusion, N omega-hydroxy-D,L-indospicine is a potent and specific inhibitor of arginase in AM phi. In cells in which, in addition to arginase, iNOS is expressed, inhibition of arginase can cause a shift of L-arginine metabolism to the NOS pathway. However, the extent of this shift appears to depend in a complex manner on the level of iNOS.1. Alveolar macrophages (AM phi) 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 omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine) might also inhibit arginase in AM phi and whether inhibition of arginase might affect L-arginine utilization by iNOS. 2. AM phi obtained by broncho-alveolar lavage of rat and rabbit isolated lungs were disseminated (2.5 or 3 x 10(6) cells per well) and allowed to adhere for 2 h. Thereafter, they were either used to study [3H]-L-arginine uptake (37 kBq, 0.1 microM, 2 min) or cultured for 20 h in the absence or presence of bacterial lipopolysaccharide (LPS). Cultured AM phi were incubated for 1 h with [3H]-L-arginine (37 kBq, 0.1 microM) and the accumulation of [3H]-L-citrulline (NOS activity) and [3H]-L-ornithine (arginase activity) was determined. 3. During 1 h incubation of rabbit AM phi with [3H]-L-arginine, no [3H]-L-citrulline, but significant amounts of [3H]-L-ornithine (150 d.p.m x 1000) were formed. N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine, present during incubation, concentration-dependently reduced [3H]-L-ornithine formation (IC50: 2 and 45 microM, respectively). 4. N omega-hydroxy-D,L-indospicine (up to 100 microM) had no effect on [3H]-L-arginine uptake into rabbit AM phi, whereas 4-hydroxyamidino-D,L-phenylalanine caused a concentration-dependent inhibition (IC50: 300 microM). 5. Rat AM phi, cultured in the absence of LPS, formed significant amounts of [3H]-L-citrulline and [3H]-L-ornithine (133 and 212 d.p.m x 1000, respectively) when incubated for 1 h with [3H]-L-arginine. When AM phi had been cultured in the presence of 0.1 or 1 microgram ml-1 LPS, the formation of [3H]-L-citrulline was enhanced by 37 +/- 8.3 and 99 +/- 12% and that of [3H]-L-ornithine reduced by 21 +/- 8.7 and 70 +/- 2.5%, respectively. 6. In rat AM phi, cultured in the absence or presence of LPS, N omega-hydroxy-D,L-indospicine (10 and 30 microM) greatly reduced formation of [3H]-L-ornithine (by 80-95%) and this was accompanied by increased formation of [3H]-L-citrulline. However, only 20-30% of the [3H]-L-arginine not metabolized to [3H]-L-ornithine after inhibition of arginase was metabolized to [3H]-L-citrulline, when the AM phi had been cultured in the absence of LPS (i.e. low level of iNOS). On the other hand, when the AM phi had been cultured in the presence of LPS (i.e. high level of iNOS), all the [3H]-L-arginine not metabolized by the inhibited arginase was metabolized to [3H]-L-citrulline. 7. In conclusion, N omega-hydroxy-D,L-indospicine is a potent and specific inhibitor of arginase in AM phi. In cells in which, in addition to arginase, iNOS is expressed, inhibition of arginase can cause a shift of L-arginine metabolism to the NOS pathway. However, the extent of this shift appears to depend in a complex manner on the level of iNOS.