title: Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin.
abstract: Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-gamma (IFN-gamma) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, NG-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-gamma than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-gamma was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-gamma was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-gamma. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pharmacology, Rutgers University, Piscataway, NJ 08855-0789.
authors.forename: D L
authors.initials: DL
authors.lastname: Laskin
authors.forename: D E
authors.initials: DE
authors.lastname: Heck
authors.forename: C R
authors.initials: CR
authors.lastname: Gardner
authors.forename: L S
authors.initials: LS
authors.lastname: Feder
authors.forename: J D
authors.initials: JD
authors.lastname: Laskin
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D004727:Endothelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004790:Enzyme Induction; D004926:Escherichia coli; D005260:Female; D005978:Glutathione / Q000172:deficiency / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity*; D008099:Liver / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase; D051381:Rats; D017207:Rats, Sprague-Dawley; D014115:Toxemia / Q000378:metabolism / Q000473:pathology
chemical_list: D008070:Lipopolysaccharides; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D005978:Glutathione