Abstract

The current study was designed to investigate the importance of cationic amino acid transporters (CATs) for the l-arginine supply to nitric oxide (NO) synthases in mouse J774A.1 macrophages and human EA.hy926 endothelial cells. CAT-1 was expressed in both cell types, whereas CAT-2B was only expressed in activated macrophages. Apparent K _M values for transport ofl-arginine in both cell types was consistent with the expression of the system y⁺ carriers CAT-1 (and CAT-2B in macrophages). In addition, l-arginine transport was Na⁺ independent and sensitive totrans-stimulation. A 2-h preincubation of activated macrophages in 2 mM l-lysine (which is exchanged forl-arginine by the CATs) reduced the intracellularl-arginine concentration from 2 mM to 160 μM. At the same time, nitric-oxide synthase (NOS) II activity was completely abolished. NOS II activity could be restored with extracellularl-arginine. No difference in NO production was seen between macrophages preincubated in l-arginine-containing buffer and incubated either with or without l-arginine during the 2-min NO assay. Incubation of endothelial cells in 2 mMl-lysine for up to 24 h decreased the intracellular l-arginine concentration from 3.5 mM to about 600 μM but did not reduce the NOS III activity. Our results suggest that both activated macrophages and endothelial cells have anl-arginine pool that is not freely exchangeable with the extracellular space. This pool seems to be accessible to NOS III in endothelial cells but not to NOS II in macrophages.