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 mediators, endotoxin (LPS) and interferon gamma (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, omega-nitro-L-arginine methyl ester (LNAME) 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 microgram/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 microM nitrite at 24 hr and reached a plateau of 55 microM 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.
Cendan
J C
JC
Department of Surgery, University of Florida College of Medicine, Gainesville, 32610-00286, USA.
Topping
D L
DL
Pruitt
J
J
Snowdy
S
S
Copeland
E M
EM
Lind
D S
DS
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D001692:Biological Transport; D004305:Dose-Response Relationship, Drug; D018836:Inflammation Mediators / Q000494:pharmacology*; D007372:Interferons / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D015674:Mammary Neoplasms, Animal / Q000378:metabolism / Q000473:pathology; D051379:Mice; D019331:NG-Nitroarginine Methyl Ester; D009569:Nitric Oxide / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured
D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D009569:Nitric Oxide; D007372:Interferons; D001120:Arginine; D019331:NG-Nitroarginine Methyl Ester