Arginase modulates nitric oxide production in activated macrophages

Abstract

In macrophages and many other cell types,l-arginine is used as a substrate by both nitric oxide synthase (NOS) and arginase to produce nitric oxide (NO) and urea, respectively. Because the availability ofl-arginine is a major determinant for NO synthesis in the activated macrophage, we hypothesized that NO production may be reduced by arginase via depleting the common substrate in this cell type. To test this hypothesis, we investigated the effect of an arginase inhibitor,l-norvaline, on NO production in J774A.1 mouse macrophages activated by lipopolysaccharide (LPS, 1.0 μg/ml) for 22 h. In the absence of LPS, macrophages produced a low level of NO. In contrast, NO production from these cells was significantly increased in the presence of LPS. Increasing extracellular levels ofl-arginine (0.01–0.8 mM) produced a concomitant increase in NO production of activated macrophages. l-Norvaline (10 mM), which specifically inhibits arginase activity (i.e., reducing urea production by 50%) without altering NOS activity, enhanced NO production (by 55%) from activated macrophages. The enhancement of NO production by l-norvaline was inversely related to the extracellular level ofl-arginine. A more pronounced increase in NO production was observed at the lower level of extracellular l-arginine, i.e., a 55 vs. 28% increase for 0.05 and 0.1 mM extracellularl-arginine, respectively. When the l-arginine concentration exceeded 0.5 mM, thel-norvaline effect was abolished. These results indicate that arginase can compete with NOS for their common substrate and thus inhibit NO production. This regulatory mechanism may be particularly important when the extracellular supply ofl-arginine is limited.

The Involvement of Tyrosine Kinases, Cyclic AMP/Protein Kinase A, and p38 Mitogen-Activated Protein Kinase in IL-13-Mediated Arginase I Induction in Macrophages: Its Implications in IL-13-Inhibited Nitric Oxide Production

“…These results also suggest that the induced arginase, by inhibiting NO production from activated macrophages, may contribute at least in part to the observed anti-inflammatory effect of IL-13 in vivo (42). It is worth noting that L-norvaline specifically inhibits arginase activity at the enzyme kinetic level (15,26), and it does not affect iNOS expression, as shown in the present study.…”

“…Therefore, the regulation of L-arginine availability to iNOS by arginase might play a role in modulating NO production from macrophages. Our previous study supports this contention, because inhibition of arginase activity enhances NO production from LPS-activated macrophages (26). Recently, arginase activity has been shown to be up-regulated by endogenous factors such as TGF-␤ (21) and Th-2 cell-derived cytokines IL-4 and IL-10 (22)(23)(24)(25).…”

“…Because both arginase and iNOS exist in activated macrophages, diversion of their common substrate, L-arginine, from the iNOS pathway to the arginase pathway may afford macrophage arginase a regulatory role in NO production. This contention is supported by our previous studies showing that NO production in activated macrophages is enhanced by blocking arginase activity (26). However, whether arginase is physiologically or pathophysiologically relevant to immune responses where NO production is involved remains unclear.…”

“…Our previous studies confirmed this finding and reported that Ͼ95% of total NO released from activated macrophages was converted to nitrite in the culture medium (26). Therefore, nitrite accumulation in the supernatant of cell culture was determined by a chemiluminescence NO analyzer (Sievers Instruments, Boulder, CO) and was used as an index of NO production.…”

“…Cells were lysed by sonication at 20 kHz (Sonic & Materials, Newtown, CT) for 30 s (10 s/cycle). Arginase activity in the cell lysates was measured as described previously (26). In brief, cell lysate (50 l) was added to 50 l of Tris-HCl (50 mM; pH 7.5) containing 10 mM MnCl 2 .…”