title: Detection of protein oxidation in endothelial cells by fluorescently labelled tyramine.
abstract: BACKGROUND
  Endothelial cell injury mediated by activated polymorphonuclear leucocytes (PMN) occurs during inflammation or reperfusion after brain ischemia. Protein oxidation caused by activated PMN may lead to functional disturbances, degeneration and death of the endothelial cells. The aim of this study was to detect protein oxidation in endothelial cells induced by activated neutrophils by using a novel fluorescent probe.
  
  
  METHODS
  Protein oxidation of Human Umbilical Vein Endothelial Cells (HUVEC) in culture was investigated by a 15-min incubation with human neutrophils activated by phorbol myristate acetate (PMA) in the presence of tyramine coupled to the succinimidyl ester of (fluorescein -5 (and-6)-carboxamido) hexanoic acid. Dityrosine bond formation as reflected by the linkage of the fluorescent tyramine to proteins was determined by Western-blotting.
  
  
  RESULTS
  The oxidative burst generated by activated neutrophils induced dityrosine formation in the extracellular proteins (ECP) of HUVEC. Similar results were obtained, when horseradish peroxidase (HRP) was used for the induction of oxidative stress. However, when hydrogen peroxide (0.1 mM) was used, dityrosine formation was not detected.
  
  
  CONCLUSIONS
  Fluorescently labelled tyramine is a powerful tool for the detection of ECP oxidation in endothelial cells. As long as the oxidation by the activated neutrophils is limited to ECP, the endothelial cells may be protected by antioxidants.
authors: Department of Cellular Signalling, Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland. grzegorz@cmdik.pan.pl
authors: G A
authors: GA
authors: Czapski
authors: D
authors: D
authors: Avram
authors: K W
authors: KW
authors: Wirtz
authors: E H
authors: EH
authors: Pap
authors: J B
authors: JB
authors: Strosznajder
mesh_terms: D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000166:cytology*; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014439:Tyramine / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D013755:Tetradecanoylphorbol Acetate; D014439:Tyramine