Cytosolic phospholipase A(2)-alpha associates with plasma membrane, endoplasmic reticulum and nuclear membrane in glomerular epithelial cells

Eicosanoids mediate complement-dependent glomerular epithelial injury in ex perimental membranous nephropathy. The release of arachidonic acid from pho spholipids by cytosolic phospholipase A(2) (cPLA(2)) is the rate-limiting s tep in eicosanoid synthesis. The present study examines the association of cPLA(2) with membranes of organelles. Glomerular epithelial cells were disr upted by homogenization in Ca2+-free buffer, organelles were separated by g radient centrifugation. The distribution of cPLA(2) and organelles was anal ysed by immunoblotting with antibodies against cPLA(2) and organelle marker s, or by enzyme assay. In cells incubated with or without the Ca2+ ionophor e ionomycin plus PMA, cPLA(2) co-localized with plasma membrane, endoplasmi c reticulum and nuclei, but not with mitochondria or Golgi. A greater amoun t of cPLA(2) was associated with membranes in stimulated cells, but membran e-associated cPLA(2) was readily detectable under resting conditions. The p attern of association of cPLA(2) with membrane in cells treated with antibo dy and complement was similar to that in cells stimulated with ionomycin pl us PMA; however, complement did not enhance the membrane association of cPL A(2) protein. To determine the functional role of membrane association of c PLA(2), phospholipids were labelled with [H-3]arachidonic acid. Cells were then incubated with or without antibody and complement and were fractionate d. Complement induced a loss of radioactivity from the plasma membrane, end oplasmic reticulum and nuclei, but not from the mitochondrial fraction. Thu s the release of arachidonic acid by cPLA(2) is due to the hydrolysis of ph ospholipids at multiple subcellular membrane sites, including the endoplasm ic reticulum, plasma membrane and nucleus.