Selective Targeting of Nitric Oxide Synthase Inhibitors to System y+ in Activated Macrophages

Abstract

SUMMARY:Amino acid transport systems mediating uptake of nitric oxide (NO) synthase inhibitors were characterized in the murine macrophage cell line J774. Treatment of J774 cells with bacterial endotoxin (LPS, 1 μg ml -1 , 24 h) selectively increased the transport capacity forInhibition studies established that the cationic transport system y + mediates uptake of L-arginine, L-NMMA and N G -iminoethyl-L-ornithine (L-NIO). A neutral transporter, with low substrate specificity and insensitive to LPS, mediates uptake of L-citrulline, L-NNA and its methyl ester L-NAME. We conclude that enhanced expression of the y + transporter in LPS-stimulated macrophages (1) may facilitate the targeting of selective inhibitors of inducible NO synthase to activated cells generating NO in endotoxin shock. ________________________________________________________________________ Activation of murine macrophage J774 cells with lipopolysaccharide (LPS) results in acycloheximide-dependent induction of NO synthase and arginine transport via system y + (1-3).In activated macrophages generation of NO by inducible NO synthase (iNOS) is critically dependent on extracellular arginine (2,4,5), despite reports that activated cells contain high intracellular arginine levels (1,6). N G -derivatized analogues of L-arginine are potent inhibitors of iNOS, and their rank order of potency has been attributed to different NOS isoforms and/or their differential rates of cellular uptake (7,8). In this context, we reported previously that N G -monomethyl-L-arginine (L-methyl ester L-NAME inhibit transport of arginine into cultured porcine aortic endothelial cells via system y + (9). Recent studies of radiolabelled L-NMMA and arginine uptake in porcine aortic endothelial cells have confirmed these findings (10).

Inhibition of nitric oxide synthesis by NG‐nitro‐L‐arginine methyl ester (L‐NAME): requirement for bioactivation to the free acid, NG‐nitro‐L‐arginine

“…According to a recent study of Forray et al (1995), a similar transporter may be responsible for uptake of L-NOARG into human erythrocytes, but the transport system for L-NAME has not been identified yet. While we observed no interference of L-NAME with any of the known amino acid transport systems in several cell types 1994;, others have found that J774 macrophages take up both L-NOARG and L-NAME via a non-selective neutral amino acid transporter (Baydoun & Mann, 1994). Definitive conclusions await uptake studies in which radiolabelled L-NAME is used.…”

Nitric oxide regulates nucleoside transport in activated B lymphocytes

“…Inhibition of NO production can be achieved either by blocking NOS activity or by limiting substrate availability. Arginine supply to the cell can be reduced either using some of the NOS inhibitors known to interact with the carrier [29,30] or by arginine depletion, which can also be achieved by different means. In a preliminary assay, Raji cells were incubated for 24 h in arginine-free E-199 medium.…”

Arginine metabolism: nitric oxide and beyond

“…For example, arginine uptake can be competitively inhibited by lysine, ornithine, canavanine and certain NOS inhibitors, including N G -monomethyl--arginine and N G -iminoethyl--ornithine, but not by other NOS inhibitors such as aminoguanidine, N G -nitro--arginine and N G -nitro--arginine methyl ester [170][171][172][173][174][175]. Thus use of NOS inhibitors that are taken up via system y + may limit the availability of arginine for other enzymes that utilize this amino acid.…”