ULTRASTRUCTURAL-LOCALIZATION OF CPLA(2) IN UNSTIMULATED AND EGF A23187-STIMULATED FIBROBLASTS/

The 85 kDa cytosolic phospholipase Az is the key enzyme in the release of arachidonic acid, To gain insight into cytosolic phospholipase A(2 ) action in mitogen-activated cells, the localization of the phospholi pase was investigated in fibroblasts upon stimulation with epidermal g rowth factor and the calcium ionophore A23187, By the use of indirect immunofluorescence microscopy, staining of endogenous cytosolic phosph olipase A(2) resulted in a punctate labeling pattern randomly distribu ted throughout the cytoplasm of the cell, Immunogold electron microsco py revealed that this punctate labeling pattern exhibited the presence of the 85 kDa phospholipase A(2) in small clusters. These clusters we re found in the cytosol in the vicinity of all organellar membranes, e xcept for the Golgi system, The enzyme showed no preference for the nu clear envelope, the endoplasmic reticulum or the plasma membrane. Stim ulation of cells with epidermal growth factor or A23187 or both did no t change the punctate immunofluorescence labeling pattern. Furthermore , a similar labeling pattern was observed by the artificial introducti on of extremely low or high intracellular calcium concentrations, Even by electron microscopy, translocation of cytosolic phospholipase A(2) to membranes was not observed after stimulation of cells,vith epiderm al growth factor and A23187. From these results it is concluded that c ytosolic phospholipase Az is localized in clusters close to membranes in stimulated as well as unstimulated fibroblasts, without preference for a specific organellar membrane.