title: Endotoxin-mediated synthesis of nitric oxide is dependent on Gq protein signal transduction.
abstract: BACKGROUND
  Nitric oxide (NO) is a ubiquitous multifunctional free radical produced during sepsis, shock, reperfusion injury, and allograft rejection. Many studies are presently evaluating the functional roles of NO production in these settings. However, the signal transduction mechanisms underlying initiation of NO production are largely unknown. This study defines the cell surface receptor proteins that mediate endotoxin-induced NO synthesis in ANA-1 murine macrophages.
  
  
  METHODS
  Endotoxin (LPS, 10 micrograms/ml) was added to ANA-1 macrophages to induce NO synthesis. In selected instances guanosine 5'-O-(2-thiodiphosphate)-trilithium salt (GOTP), pertussis toxin, cholera toxin, or suramin were added as inhibitors of specific subclasses of heterotrimeric G proteins. Calphostin was added as a protein kinase C inhibitor, and ET-OCH3 was added as a phospholipase C-beta inhibitor. NO release was quantified by measurement of the NO metabolite, nitrite. Membrane guanosine triphosphatase (GTPase) activity was also analyzed. Steady-state levels of inducible nitric oxide synthase (iNOS) mRNA were determined by using reverse transcription-polymerase chain reaction analysis.
  
  
  RESULTS
  Inhibition of G protein function by suramin or GOTP significantly decreased synthesis of NO and expression of iNOS mRNA. Pertussis and cholera toxin did not alter NO synthesis, suggesting that the Gi and Gs classes are not involved. Inhibition of protein kinase C or upstream phospholipase C-beta activity decreased NO synthesis, implicating the Gq class of heterotrimeric G proteins.
  
  
  CONCLUSIONS
  In ANA-1 macrophages, endotoxin-mediated NO synthesis is dependent on heterotrimeric Gq protein-phospholipase C-beta-protein kinase C signal transduction.
authors.affiliation: Department of Surgery, University of Maryland Medical System, Baltimore, USA.
authors.forename: P C
authors.initials: PC
authors.lastname: Kuo
authors.forename: R A
authors.initials: RA
authors.lastname: Schroeder
authors.forename: S T
authors.initials: ST
authors.lastname: Bartlett
mesh_terms: D000818:Animals; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D004731:Endotoxins / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D019204:GTP-Binding Proteins / Q000378:metabolism*; D019833:Genistein; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages; D051379:Mice; D009281:Naphthalenes / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D037342:Pertussis Toxin; D010742:Phospholipid Ethers / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013498:Suramin / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D004731:Endotoxins; D004791:Enzyme Inhibitors; D007529:Isoflavones; D008070:Lipopolysaccharides; D009281:Naphthalenes; D010742:Phospholipid Ethers; D012333:RNA, Messenger; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; C061465:calphostin complex; D006153:Guanosine Diphosphate; C026659:edelfosine; D009569:Nitric Oxide; D013498:Suramin; C023427:guanosine 5'-O-(2-thiodiphosphate); D002772:Cholera Toxin; D019833:Genistein; D019001:Nitric Oxide Synthase; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate