Liu J, Takano T, Papillon J, Khadir A, Cybulsky A V
Departments of Physiology and Medicine, McGill University, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec H3A 1A1, Canada.
Biochem J. 2001 Jan 1;353(Pt 1):79-90.
Eicosanoids mediate complement-dependent glomerular epithelial injury in experimental membranous nephropathy. The release of arachidonic acid from phospholipids by cytosolic phospholipase A(2) (cPLA(2)) is the rate-limiting step in eicosanoid synthesis. The present study examines the association of cPLA(2) with membranes of organelles. Glomerular epithelial cells were disrupted by homogenization in Ca(2+)-free buffer; organelles were separated by gradient centrifugation. The distribution of cPLA(2) and organelles was analysed by immunoblotting with antibodies against cPLA(2) and organelle markers, or by enzyme assay. In cells incubated with or without the Ca(2+) ionophore ionomycin plus PMA, cPLA(2) co-localized with plasma membrane, endoplasmic reticulum and nuclei, but not with mitochondria or Golgi. A greater amount of cPLA(2) was associated with membranes in stimulated cells, but membrane-associated cPLA(2) was readily detectable under resting conditions. The pattern of association of cPLA(2) with membrane in cells treated with antibody and complement was similar to that in cells stimulated with ionomycin plus PMA; however, complement did not enhance the membrane association of cPLA(2) protein. To determine the functional role of membrane association of cPLA(2), phospholipids were labelled with [(3)H]arachidonic acid. Cells were then incubated with or without antibody and complement and were fractionated. Complement induced a loss of radioactivity from the plasma membrane, endoplasmic reticulum and nuclei, but not from the mitochondrial fraction. Thus the release of arachidonic acid by cPLA(2) is due to the hydrolysis of phospholipids at multiple subcellular membrane sites, including the endoplasmic reticulum, plasma membrane and nucleus.
类二十烷酸介导实验性膜性肾病中补体依赖性肾小球上皮损伤。胞质磷脂酶A2（cPLA2）从磷脂中释放花生四烯酸是类二十烷酸合成的限速步骤。本研究检测cPLA2与细胞器膜的关联。肾小球上皮细胞在无钙缓冲液中匀浆破碎；细胞器通过梯度离心分离。通过用抗cPLA2和细胞器标志物的抗体进行免疫印迹或酶测定来分析cPLA2和细胞器的分布。在用或不用钙离子载体离子霉素加佛波酯（PMA）孵育的细胞中，cPLA2与质膜、内质网和细胞核共定位，但不与线粒体或高尔基体共定位。在受刺激的细胞中，有更多的cPLA2与膜相关联，但在静息条件下也能轻易检测到膜相关的cPLA2。用抗体和补体处理的细胞中，cPLA2与膜的关联模式与用离子霉素加PMA刺激的细胞相似；然而，补体并未增强cPLA2蛋白与膜的关联。为了确定cPLA2与膜关联的功能作用，用[3H]花生四烯酸标记磷脂。然后将细胞用或不用抗体和补体孵育并进行分级分离。补体导致质膜、内质网和细胞核中的放射性损失，但线粒体部分没有。因此，cPLA2介导的花生四烯酸释放是由于包括内质网、质膜和细胞核在内的多个亚细胞膜位点的磷脂水解所致。
