title: LPS-stimulated RAW264.7 macrophage CAT-2-mediated l-arginine uptake and nitric oxide biosynthesis is inhibited by omega fatty acid lipid emulsion.
abstract: BACKGROUND
  Omega-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.
  
  
  METHODS
  Confluent 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 [(3)H]-l-arginine. The expression of iNOS and CAT-2 mRNA in harvested RAW264.7 was quantified by reverse transcriptase-polymerase chain reaction.
  
  
  RESULTS
  NO 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).
  
  
  CONCLUSIONS
  These 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.
authors.affiliation: Research Institute of General Surgery, Jinling Hospital, Nanjing, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Huang
authors.forename: Chuanjiang
authors.initials: C
authors.lastname: Huang
authors.forename: Yunzhao
authors.initials: Y
authors.lastname: Zhao
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.forename: Jianan
authors.initials: J
authors.lastname: Ren
authors.forename: Ning
authors.initials: N
authors.lastname: Li
authors.forename: Jieshou
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D027202:Cationic Amino Acid Transporter 2 / Q000378:metabolism*; D002460:Cell Line; D004655:Emulsions; D015525:Fatty Acids, Omega-3 / Q000494:pharmacology*; D043371:Fatty Acids, Omega-6 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D027202:Cationic Amino Acid Transporter 2; D004655:Emulsions; D015525:Fatty Acids, Omega-3; D043371:Fatty Acids, Omega-6; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D009569:Nitric Oxide; D001120:Arginine; D052247:Nitric Oxide Synthase Type II