title: A role for heterotrimeric GTP-binding proteins and ERK1/2 in insulin-mediated, nitric-oxide-dependent, cyclic GMP production in human umbilical vein endothelial cells.
abstract: OBJECTIVE
  Insulin is known to stimulate endothelial nitric oxide synthesis, although much remains unknown about the intracellular mechanisms involved. This study aims to examine, in human endothelial cells, the specific contribution of heterotrimeric Gi proteins and extracellular signal-regulated protein kinases 1/2 (ERK1/2) in insulin signalling upstream of nitric-oxide-dependent cyclic GMP production.
  
  
  METHODS
  Human umbilical vein endothelial cells were treated with 1 nmol/l insulin in the presence or absence of inhibitors of tyrosine kinases (erbstatin), Gi proteins (pertussis toxin) or ERK1/2 (PD098059 or U0126), and nitric oxide production was examined by quantification of intracellular cyclic GMP. Activation/phosphorylation of ERK1/2 by insulin was examined by immunoblotting with specific antibodies, and direct association of the insulin receptor with Gi proteins was examined by immunoprecipitation.
  
  
  RESULTS
  Treatment of cells with a physiological concentration of insulin (1 nmol/l) for 5 min increased nitric-oxide-dependent cyclic GMP accumulation by 3.3-fold, and this was significantly inhibited by erbstatin. Insulin-stimulated cyclic GMP production was significantly reduced by pertussis toxin and by the inhibitors of ERK1/2, PD098059 and U0126. Immunoblotting indicated that insulin stimulated the phosphorylation of ERK1/2 after 5 min and 1 h, and that this was completely abolished by pertussis toxin, but insensitive to the nitric oxide synthase inhibitor L-NAME. No direct interaction of the insulin receptor beta with Gialpha2 could be demonstrated by immunoprecipitation.
  
  
  CONCLUSIONS
  This study demonstrates, for the first time, that nitric oxide production induced by physiologically relevant concentrations of insulin, is mediated by the post-receptor activation of a pertussis-sensitive GTP-binding protein and subsequent downstream activation of the ERK1/2 cascade.
authors.affiliation: Institute for Biomedical and Clinical Science, Peninsula Medical School, Exeter, EX1 2LU, UK. jackie.whatmore@pms.ac.uk
authors.forename: O
authors.initials: O
authors.lastname: Konopatskaya
authors.forename: A C
authors.initials: AC
authors.lastname: Shore
authors.forename: J E
authors.initials: JE
authors.lastname: Tooke
authors.forename: J L
authors.initials: JL
authors.lastname: Whatmore
mesh_terms: D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006131:Growth Inhibitors / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D037342:Pertussis Toxin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000502:physiology*
chemical_list: D004791:Enzyme Inhibitors; D006131:Growth Inhibitors; D006133:Growth Substances; D006873:Hydroquinones; D007328:Insulin; D009569:Nitric Oxide; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020962:Heterotrimeric GTP-Binding Proteins; D006152:Cyclic GMP; C048212:erbstatin
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