Cytosolic phospholipase A2-alpha and cyclooxygenase-2 localize to intracellular membranes of EA.hy.926 endothelial cells that are distinct from the endoplasmic reticulum and the Golgi apparatus.

Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme that plays an important role in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid can be converted subsequently into prostacyclin, a potent vasodilator and inhibitor of platelet activation, through the action of cyclooxygenase (COX) enzymes. Here we study the relocation of cPLA2-alpha in human EA.hy.926 endothelial cells following stimulation with the calcium-mobilizing agonist, A23187. Relocation of cPLA2-alpha was seen to be highly cell specific, and in EA.hy.926 cells occurred primarily to intracellular structures resembling the endoplasmic reticulum (ER) and Golgi. In addition, relocation to both the inner and outer surfaces of the nuclear membrane was observed. Colocalization studies with markers for these subcellular organelles, however, showed colocalization of cPLA2-alpha with nuclear membrane markers but not with ER or Golgi markers, suggesting that the relocation of cPLA2-alpha occurs to sites that are separate from these organelles. Colocalization with annexin V was also observed at the nuclear envelope, however, little overlap with staining patterns for the potential cPLA2-alpha interacting proteins, annexin I, vimentin, p11 or actin, was seen in this cell type. In contrast, cPLA2-alpha was seen to partially colocalize specifically with the COX-2 isoform at the ER-resembling structures, but not with COX-1. These studies suggest that cPLA2-alpha and COX-2 may function together at a distinct and novel compartment for eicosanoid signalling.