Substrate supply for nitric-oxide synthase in macrophages and endothelial cells: role of cationic amino acid transporters.
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 of L-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 to trans-stimulation. A 2-h preincubation of activated macrophages in 2 mM L-lysine (which is exchanged for L-arginine by the CATs) reduced the intracellular L-arginine concentration from 2 mM to 160 microM. At the same time, nitric-oxide synthase (NOS) II activity was completely abolished. NOS II activity could be restored with extracellular L-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 mM L-lysine for up to 24 h decreased the intracellular L-arginine concentration from 3.5 mM to about 600 microM but did not reduce the NOS III activity. Our results suggest that both activated macrophages and endothelial cells have an L-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.
Closs
E I
EI
Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany. Closs@mail.uni-mainz.de
Scheld
J S
JS
Sharafi
M
M
Förstermann
U
U
D026905:Amino Acid Transport Systems; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D051381:Rats; D013379:Substrate Specificity
D026905:Amino Acid Transport Systems; D002352:Carrier Proteins; D008070:Lipopolysaccharides; D009569:Nitric Oxide; D001120:Arginine; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat; C496315:Nos3 protein, mouse; C496316:Nos3 protein, rat