Ma Z, Ramanadham S, Wohltmann M, Bohrer A, Hsu F F, Turk J
Mass Spectrometry Resource, Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2001 Apr 20;276(16):13198-208. doi: 10.1074/jbc.M010423200. Epub 2001 Jan 22.
A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.
一种胞质84 kDa的ⅥA组磷脂酶A2（iPLA2β）已从多个来源克隆得到，其催化作用不需要Ca2+，这些来源包括大鼠和人胰岛β细胞以及小鼠P388D1细胞。人们提出了许多iPLA2β的潜在功能，包括在β细胞胰岛素分泌中的信号传导作用以及在生成溶血磷脂酰胆碱受体以促进花生四烯酸掺入P388D1细胞磷脂酰胆碱（PC）中的作用。关于iPLA2β功能的提议部分基于用溴代烯醇内酯（BEL）自杀底物抑制iPLA2β活性的效果，但BEL也抑制磷脂酸磷酸水解酶-1和ⅥB组磷脂酶A2。通过分子生物学手段操纵iPLA2β表达是研究iPLA2β功能的另一种方法，我们使用含有iPLA2β cDNA的逆转录病毒构建体制备了两个稳定过表达iPLA2β的INS-1胰岛素瘤细胞克隆系。与亲本INS-1细胞或用空载体转染的细胞相比，两个过表达iPLA2β的细胞系对葡萄糖和升高cAMP的试剂均表现出增强的胰岛素分泌反应，并且BEL可显著减弱刺激后的分泌。对花生四烯酸掺入INS-1细胞PC的电喷雾电离质谱分析表明，iPLA2β的过表达或抑制均不影响INS-1细胞中该过程的速率或程度。用针对iPLA2β的抗体进行的免疫细胞荧光研究表明，升高cAMP的试剂会增加INS-1细胞中核周荧光，这表明iPLA2β与细胞核相关联。这些研究更符合iPLA2β在胰岛素分泌β细胞中起信号传导作用而非管家作用的观点。
