Calcium-independent phospholipases A2 in murine osteoblastic cells and their inhibition by bromoenol lactone: impact on arachidonate dynamics and prostaglandin synthesis |
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| Authors: | Hans Jörg Leis Werner Windischhofer |
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| Institution: | 1. 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, Austriahans.leis@medunigraz.at;3. 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 |
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| Abstract: | Context: 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: 14C-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.Discussion: 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. Conclusion: BEL must be used with caution in a cellular environment due to conditions of extreme oxidative stress. |
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| Keywords: | Arachidonate release bromoenol lactone iPLA2 osteoblast prostaglandin |
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