Identification of calcium-independent phospholipase A2 (iPLA2) beta, and not iPLA2gamma, 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, beta-cells, and neurons) has been demonstrated to be primarily mediated by calcium-independent phospholipases A(2) (iPLA(2)s) that are inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Recently, the family of mammalian iPLA(2)s has been extended to include iPLA(2)gamma, which previously could not be pharmacologically distinguished from iPLA(2)beta. To determine whether iPLA(2)beta or iPLA(2)gamma (or both) were the enzymes responsible for arginine vasopressin (AVP)-induced AA release from A-10 cells, it became necessary to inhibit selectively iPLA(2)beta and iPLA(2)gamma in intact cells. We hypothesized that the R- and S-enantiomers of BEL would possess different inhibitory potencies for iPLA(2)beta and iPLA(2)gamma. 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 iPLA(2)beta in comparison to iPLA(2)gamma. Conversely, (R)-BEL was approximately an order of magnitude more selective for iPLA(2)gamma than iPLA(2)beta. The AVP-induced liberation of AA from A-10 cells was selectively inhibited by (S)-BEL (IC(50) approximately 2 microm) but not (R)-BEL, demonstrating that the overwhelming majority of AA release is because of iPLA(2)beta and not iPLA(2)gamma 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 iPLA(2)beta in AVP-induced AA release.
Jenkins
Christopher M
CM
Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Han
Xianlin
X
Mancuso
David J
DJ
Gross
Richard W
RW
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001127:Arginine Vasopressin / Q000494:pharmacology*; D002460:Cell Line; D004789:Enzyme Activation; D054522:Group VI Phospholipases A2; D007700:Kinetics; D020935:MAP Kinase Signaling System; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010741:Phospholipases A / Q000737:chemistry / Q000502:physiology*; D054467:Phospholipases A2; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport; D011753:Pyrones / Q000494:pharmacology; D051381:Rats; D013237:Stereoisomerism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014667:Vasopressins / Q000494:pharmacology
D009281:Naphthalenes; D010726:Phosphodiesterase Inhibitors; D011753:Pyrones; D014667:Vasopressins; D001127:Arginine Vasopressin; D016718:Arachidonic Acid; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D011493:Protein Kinase C; D010741:Phospholipases A; D054522:Group VI Phospholipases A2; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate