Inflammatory mediators stimulate arginine transport and arginine-derived nitric oxide production in a murine breast cancer cell line

Inflammatory mediators stimulate arginine-derived nitric oxide (NO) production in a variety of cells. The purpose of this study was to determine if the inflammatory madiators, endotoxin (LPS) and interferon γ (IFN), stimulate arginine transport and nitric oxide production in a murine breast cancer cell line. We also investigated the effect of the nitric oxide synthase (NOS) inhibitors, ω-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (AG), as well as the effect of varying the concentration of L- arginine in the cellular media, on arginine transport and NO production in these tumors cells. Confluent EMT-6 murine breast cancer cells were incubated with LPS (10 μg/ml) and IFN (50 units/ml) in the presence or absence of the NOS inhibitors, L-NAME (2 mM) or AG (1 mM), and arginine transport (using L- [³H]arginine) and NO production (the stable end-product nitrite was assayed using the Greiss reagent) were measured at various time points. In addition, the effect of varying the concentration of L-arginine (0, 10, 100, 1000, 10,000 mM) in the cellular media on stimulated L-arginine transport and nitrite accumulation was assessed. Incubation of EMT-6 with LPS and IFN stimulated arginine transport approximately 70% over control levels at 12 hr and transport returned to basal levels at 24 hr. LPS/IFN-stimulated EMT-6 cells produced 25 μM nitrite at 24 hr and reached a plateau of 55 μM nitrite at 48 hr. The NO synthase inhibitors, L-NAME and AG, failed to inhibit basal and stimulated levels of arginine transport, but significantly inhibited nitrite accumulation, which was restored by 10 mM L-arginine. Finally, L-arginine was necessary in the media for nitrite accumulation by LPS/IFN-stimulated cells, with maximal accumulation at 1 mM L-arginine. In summary, LPS/IFN stimulate arginine transport and NO production in the EMT-6 breast cancer cell line. L-NAME and AG do not inhibit basal or stimulated arginine transport in this tumor cell line and extracellular L-arginine is required for NO synthesis in these cells. LPS/IFN stimulation of arginine transport may represent an adaptive response to provide increased substrate for enhanced tumor cell NO production.