title: Calcium-independent phospholipases A2 in murine osteoblastic cells and their inhibition by bromoenol lactone: impact on arachidonate dynamics and prostaglandin synthesis.
abstract: BACKGROUND
  Bromoenol lactone (BEL) is an inhibitor of group VI phospholipases (iPLA2s), but has been shown to have severe side effects.
  
  
  OBJECTIVE
  iPLA2 characterization in osteoblasts and effect of BEL on prostaglandin (PG) E2 formation.
  
  
  METHODS
  iPLA2 expression: RT-PCR, Western Blotting. PGE2 formation: GC-MS after stimulation, treatment with inhibitors or gene silencing. Arachidonate (AA) reacylation into phospholipids, inhibitor reaction products, PGHS-1 modification proteomic analysis: HR-LC-MS/MS. AA accumulation: (14)C-AA.
  
  
  RESULTS
  iPLA2ß and iPLA2γ were expressed and functionally active. BEL inhibition up to 20 μM caused AA accumulation and enhanced PGE2 formation, followed by a decrease at higher concentrations. BEL reacted with intracellular cysteine and GSH leading to GSH depletion and oxidative stress.
  
  
  CONCLUSIONS
  Initial PGE2 enhancement after BEL inhibition is due to iPLA2-independent accumulation of AA. GSH depletion caused by high BEL concentrations is responsible for the decrease in PGE2 production.
  
  
  CONCLUSIONS
  BEL must be used with caution in a cellular environment due to conditions of extreme oxidative stress.
authors.affiliation: a University Hospital of Youth and Adolescence Medicine, Medical University of Graz, Research Unit of Analytical Mass Spectrometry, Cell Biology and Biochemistry of Inborn Errors of Metabolism , Graz , Austria.
authors.forename: Hans Jörg
authors.initials: HJ
authors.lastname: Leis
authors.affiliation: a University Hospital of Youth and Adolescence Medicine, Medical University of Graz, Research Unit of Analytical Mass Spectrometry, Cell Biology and Biochemistry of Inborn Errors of Metabolism , Graz , Austria.
authors.forename: Werner
authors.initials: W
authors.lastname: Windischhofer
mesh_terms: D016475:3T3 Cells; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D051379:Mice; D009281:Naphthalenes / Q000494:pharmacology*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D054512:Phospholipases A2, Calcium-Independent / Q000378:metabolism*; D011453:Prostaglandins / Q000096:biosynthesis*; D011753:Pyrones / Q000494:pharmacology*
chemical_list: D009281:Naphthalenes; D011453:Prostaglandins; D011753:Pyrones; D016718:Arachidonic Acid; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D054512:Phospholipases A2, Calcium-Independent
keywords: Arachidonate release; bromoenol lactone; iPLA2; osteoblast; prostaglandin