Inhibition of nitric oxide synthase abrogates lipopolysaccharides-induced up-regulation of L-arginine uptake in rat alveolar macrophages.

It was tested whether the inducible nitric oxide synthase (iNOS) pathway might be involved in lipopolysaccharides-(LPS)-induced up-regulation of L-arginine transport in rat alveolar macrophages (AM). AM were cultured in absence or presence of LPS. Nitrite accumulation was determined in culture media and cells were used to study [3H]-L-arginine uptake or to isolate RNA for RT - PCR. Culture in presence of LPS (1 microg ml(-1), 20 h) caused 11 fold increase of nitrite accumulation and 2.5 fold increase of [3H]-L-arginine uptake. The inducible NO synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) present alone during culture had only marginal effects on [3H]-L-arginine uptake. However, AMT present during culture additionally to LPS, suppressed LPS-induced nitrite accumulation and LPS-stimulated [3H]-L-arginine uptake in the same concentration-dependent manner. AMT present only for the last 30 min of the culture period had similar effects on [3H]-L-arginine uptake. AMT present only during the uptake period also inhibited LPS-stimulated [3H]-L-arginine uptake, but with lower potency. The inhibitory effect of AMT could not be opposed by the NO releasing compound DETA NONOate. LPS caused an up-regulation of the mRNA for the cationic amino acid transporter CAT-2B, and this effect was not affected by AMT. AMT (100 microM) did not affect L-arginine transport studied by electrophysiological techniques in Xenopus laevis oocytes expressing either the human cationic amino acid transporter hCAT-1 or hCAT-2B. In conclusion, iNOS inhibition in rat AM abolished LPS-activated L-arginine uptake. This effect appears to be caused by reduced flow of L-arginine through the iNOS pathway.

Activation of L-arginine transport by protein kinase C in rabbit, rat and mouse alveolar macrophages.

1. The role of protein kinase C in controlling L-arginine transport in alveolar macrophages was investigated. 2. L-[3H]Arginine uptake in rabbit alveolar macrophages declined by 80 % after 20 h in culture. 4beta-Phorbol 12-myristate 13-acetate (PMA), but not 4alpha-phorbol 12-myristate 13-acetate (alpha-PMA), present during 20 h culture, enhanced L-[3H]arginine uptake more than 10-fold. Staurosporine and chelerythrine opposed this effect. 3. L-[3H]Arginine uptake was saturable and blockable by L-lysine. After PMA treatment Vmax was increased more than 5-fold and Km was reduced from 0.65 to 0.32 mM. 4. Time course experiments showed that PMA increased L-[3H]arginine uptake almost maximally within 2 h. This short-term effect was not affected by cycloheximide or actinomycin D. 5. L-[3H]Arginine uptake and its stimulation by PMA was also observed in sodium-free medium. 6. L-Leucine (0.1 mM) inhibited L-[3H]arginine uptake by 50 % in sodium-containing medium, but not in sodium-free medium. At 1 mM, L-leucine caused significant inhibition in sodium-free medium also. L-Leucine showed similar effects on PMA-treated cells. 7. N-Ethylmaleimide (200 microM, 10 min) reduced L-[3H]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 L-arginine uptake, which is markedly stimulated by protein kinase C, probably by modulation of the activity of already expressed cationic amino acid transporters.