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

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.