LPS-stimulated RAW264.7 macrophage CAT-2–mediated l-arginine uptake and nitric oxide biosynthesis is inhibited by omega fatty acid lipid emulsion

Abstract

BackgroundOmega-3 fatty acid (ω-3 FA) lipid emulsion has been reported to inhibit nitric oxide (NO) production and alter inducible nitric oxide synthase (iNOS) protein expression in lipopolysaccharide (LPS)-stimulated murine macrophages. However, the role of cellular uptake of l-arginine and iNOS transcription in ω-3 FA emulsion-induced inhibition of NO has not been explored. In addition, cationic amino acid transporter-2 (CAT-2) can regulate iNOS activity. The effect of ω-3 FA emulsion on CAT-2 expression is unknown. In the present study, we hypothesized that ω-3 FA emulsion pretreatment would decrease the production of NO in LPS-stimulated macrophages and that this effect would occur through alterations in the cellular uptake of l-arginine and CAT-2 expression, in addition to iNOS expression. MethodsConfluent immortalized murine macrophages (RAW264.7cells) were incubated with Dulbecco’s modified Eagle’s medium, ω-3 FA emulsion, or an isoenergetic ω-6 lipid emulsion for 4 h. The cells were washed and then stimulated with LPS (1 μg/mL) or media alone for 12 or 24 h before harvesting. Greiss reagent was used to assess NO production of plate well supernatants. Cellular uptake of l-arginine was assessed through [3H]-l-arginine. The expression of iNOS and CAT-2 mRNA in harvested RAW264.7 was quantified by reverse transcriptase-polymerase chain reaction. ResultsNO production of unstimulated RAW264.7 cells was similar in all groups. After LPS stimulation, ω-3 FA pretreatment at 12 and 24 h produced significantly less NO (P < 0.05) compared with ω-6 FA or media only. ω-3 FA pretreatment at 12 and 24 h resulted in less l-arginine uptake. iNOS and CAT-2 mRNA was significantly decreased with ω-3 FA pretreatment compared with ω-6 FA or media-only treatment (P < 0.05). ConclusionsThese experiments demonstrated that, in addition to other anti-inflammatory effects, ω-3 FA lipid emulsion also significantly lowers NO production and l-arginine transport through altered expression of iNOS and CAT-2 in LPS-stimulated RAW264.7 macrophage cells.