Identification of calcium-independent phospholipase A2 (iPLA2) β, and not iPLA2γ, as the mediator of arginine vasopressin-induced arachidonic acid release in A-10 smooth muscle cells. Enantioselective mechanism-based discrimination of mammalian iPLA2s

Christopher M. Jenkins, Xianlin Han, David J. Mancuso, Richard W. Gross

Research output: Contribution to journalArticle

150 Scopus citations

Abstract

The agonist-stimulated release of arachidonic acid (AA) from cellular phospholipids in many cell types (e.g. myocytes, β-cells, and neurons) has been demonstrated to be primarily mediated by calcium-independent phospholipases A2 (iPLA2s) that are inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Recently, the family of mammalian iPLA2s has been extended to include iPLA2γ, which previously could not be pharmacologically distinguished from iPLA2β. To determine whether iPLA2β or iPLA2γ (or both) were the enzymes responsible for arginine vasopressin (AVP)-induced AA release from A-10 cells, it became necessary to inhibit selectively iPLA2β and iPLA2γ in intact cells. We hypothesized that the R- and S-enantiomers of BEL would possess different inhibitory potencies for iPLA2β and iPLA2γ. Accordingly, racemic BEL was separated into its enantiomeric constituents by chiral high pressure liquid chromatography. Remarkably, (S)-BEL was approximately an order of magnitude more selective for iPLA2β in comparison to iPLA2γ. Conversely, (R)-BEL was approximately an order of magnitude more selective for iPLA2γ than iPLA2β. The AVP-induced liberation of AA from A-10 cells was selectively inhibited by (S)-BEL (IC50 ∼2 μM) but not (R)-BEL, demonstrating that the overwhelming majority of AA release is because of iPLA2β and not iPLA2γ activity. Furthermore, pretreatment of A-10 cells with (S)-BEL did not prevent AVP-induced MAPK phosphorylation or protein kinase C translocation. Finally, two different cell-permeable protein kinase C activators (phorbol-12-myristate-13-acetate and 1,2-dioctanoyl-sn-glycerol) could not restore the ability of A-10 cells to release AA after exposure to (S)-BEL, thus supporting the downstream role of iPLA2β in AVP-induced AA release.

Original languageEnglish
Pages (from-to)32807-32814
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number36
DOIs
StatePublished - Sep 6 2002

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