Jenkins Christopher M, Han Xianlin, Mancuso David J, Gross Richard W
Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2002 Sep 6;277(36):32807-14. doi: 10.1074/jbc.M202568200. Epub 2002 Jun 27.
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.
在许多细胞类型（如心肌细胞、β细胞和神经元）中，激动剂刺激细胞磷脂释放花生四烯酸（AA）的过程主要由不依赖钙的磷脂酶A2（iPLA2）介导，该酶可被基于机制的抑制剂（E）-6-（溴亚甲基）-3-（1-萘基）-2H-四氢吡喃-2-酮（BEL）抑制。最近，哺乳动物iPLA2家族已扩展至包括iPLA2γ，此前在药理学上无法将其与iPLA2β区分开来。为了确定iPLA2β或iPLA2γ（或两者）是否是负责精氨酸加压素（AVP）诱导A-10细胞释放AA的酶，有必要在完整细胞中选择性抑制iPLA2β和iPLA2γ。我们推测BEL的R-和S-对映体对iPLA2β和iPLA2γ具有不同的抑制效力。因此，通过手性高压液相色谱将外消旋BEL分离为其对映体成分。值得注意的是，与iPLA2γ相比，（S）-BEL对iPLA2β的选择性高约一个数量级。相反，（R）-BEL对iPLA2γ的选择性比对iPLA2β高约一个数量级。（S）-BEL（IC50约为2微摩尔）可选择性抑制AVP诱导的A-10细胞释放AA，但（R）-BEL则不能，这表明绝大多数AA释放是由于iPLA2β的活性而非iPLA2γ的活性。此外，用（S）-BEL预处理A-10细胞并不能阻止AVP诱导的MAPK磷酸化或蛋白激酶C易位。最后，两种不同的细胞可渗透蛋白激酶C激活剂（佛波醇-12-肉豆蔻酸酯-13-乙酸酯和1,2-二辛酰基-sn-甘油）在A-10细胞暴露于（S）-BEL后不能恢复其释放AA的能力，从而支持了iPLA2β在AVP诱导的AA释放中的下游作用。
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