title: Environmental pH regulates LPS-induced nitric oxide formation in murine macrophages.
abstract: This study was designed to determine how pH affects nitric oxide (NO) formation induced by lipopolysaccharide (LPS) in cultured murine macrophages (RAW 264.7). The initial pH of LPS-containing culture media was adjusted to one of eight values (6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, and 8.2). After exposure to LPS for eighteen hours, the cultures were harvested for analysis of mRNA, protein, and nitrate/nitrite (stable by-products of NO). Analyses for these substances were performed using semiquantitative RT-PCR, immunoblotting, and colorimetric Griess assays, respectively. We found that acidic culture media favored expression of inducible nitric oxide synthase (iNOS) mRNA. However, alkaline media favored expression of iNOS protein. Our findings suggest that post-transcriptional mechanisms predominate over transcriptional ones in order to regulate pH-mediated effects on NO formation by murine macrophages. The optimal pH for NO formation by iNOS was found in our study to be around 7.2.
authors.affiliation: Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan.
authors.forename: Chun-Jen
authors.initials: CJ
authors.lastname: Huang
authors.forename: Ikram U
authors.initials: IU
authors.lastname: Haque
authors.forename: Paul N
authors.initials: PN
authors.lastname: Slovin
authors.forename: R B
authors.initials: RB
authors.lastname: Nielsen
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Fang
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Skimming
mesh_terms: D000818:Animals; D002460:Cell Line; D003470:Culture Media / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009566:Nitrates / Q000032:analysis; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000032:analysis; D011506:Proteins / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis
chemical_list: D003470:Culture Media; D008070:Lipopolysaccharides; D009566:Nitrates; D009573:Nitrites; D011506:Proteins; D012333:RNA, Messenger; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse