title: Specific inhibition of nitric oxide production in macrophages by phosphorothioate antisense oligonucleotides.
abstract: The effects of antisense oligonucleotides (ODNs) on nitric oxide (NO) production induced by lipopolysaccharide (LPS) were investigated using thioglycollate-induced mouse peritoneal macrophages. Antisense phosphorothioate ODNs (S-oligo) corresponding to a sequence in the neighborhood of the AUG initiation codon of a mouse inducible nitric oxide synthase (iNOS) mRNA, which has a G-quartet motif in its antisense sequence, inhibited NO induction in a dose-dependent manner. Antisense phosphodiester ODNs (D-oligo), 5'- and 3'-terminal phosphorothioate-modified antisense ODNs and control scramble and missense S-oligos had no such effect. In addition, control nonsense and two mismatched S-oligos, which include G-quartet motif in their sequences, inhibited NO induction to approximately 50% of those in the control. Antisense S-oligo showed the inhibitory effect on NO production by exposure of macrophages to various concentrations of LPS. Western blot analysis using anti-mouse inducible nitric oxide synthase (iNOS) antibody revealed that antisense S-oligo specifically removed an immunoreactive band at 130 kDa. In addition, the results of reverse transcription-polymerase chain reaction (RT-PCR) revealed that the antisense effect originated from a specific reduction of the targeted iNOS mRNA by hybridization with the antisense S-oligo. Furthermore, no ODNs affected beta-actin mRNA and tumor necrosis factor alpha (TNF-alpha) expression in macrophages stimulated by LPS. These findings demonstrated that antisense S-oligo inhibited NO production derived from iNOS expression in macrophages by an antisense mechanism, including the aptameric effect partially mediated by the G-quartet motif.
authors.affiliation: School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Arima
authors.forename: T
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Y
authors.initials: Y
authors.lastname: Aramaki
authors.forename: K
authors.initials: K
authors.lastname: Ishidate
authors.forename: S
authors.initials: S
authors.lastname: Tsuchiya
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D008262:Macrophage Activation / Q000187:drug effects; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D013864:Thioglycolates / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013864:Thioglycolates; D013873:Thionucleotides; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse