Eicosanoids mediate complement-dependent glomerular epithelial injury in experimental membranous nephropathy. The release of arachidonic acid from phospholipids by cytosolic phospholipase A2 (cPLA2) is the rate-limiting step in eicosanoid synthesis. The present study examines the association of cPLA2 with membranes of organelles. Glomerular epithelial cells were disrupted by homogenization in Ca2+-free buffer; organelles were separated by gradient centrifugation. The distribution of cPLA2 and organelles was analysed by immunoblotting with antibodies against cPLA2 and organelle markers, or by enzyme assay. In cells incubated with or without the Ca2+ ionophore ionomycin plus PMA, cPLA2 co-localized with plasma membrane, endoplasmic reticulum and nuclei, but not with mitochondria or Golgi. A greater amount of cPLA2 was associated with membranes in stimulated cells, but membrane-associated cPLA2 was readily detectable under resting conditions. The pattern of association of cPLA2 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 cPLA2 protein. To determine the functional role of membrane association of cPLA2, phospholipids were labelled with [3H]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 cPLA2 is due to the hydrolysis of phospholipids at multiple subcellular membrane sites, including the endoplasmic reticulum, plasma membrane and nucleus.
Cytosolic phospholipase A2-α associates with plasma membrane, endoplasmic reticulum and nuclear membrane in glomerular epithelial cells
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Jianhong LIU, Tomoko TAKANO, Joan PAPILLON, Abdelkrim KHADIR, Andrey V. CYBULSKY; Cytosolic phospholipase A2-α associates with plasma membrane, endoplasmic reticulum and nuclear membrane in glomerular epithelial cells. Biochem J 1 January 2001; 353 (1): 79–90. doi: https://doi.org/10.1042/bj3530079
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