Critical duration of intracellular Ca2+ response required for continuous translocation and activation of cytosolic phospholipase A2

When cells are exposed to certain external stimuli, arachidonic acid (AA) is released from the membrane and serves as a precursor of various types of eicosanoids. A Ca2+-regulated cytosolic phospholipase A(2) (cPLA(2)) plays a dominant role in the release of AA. To closely examine the relation between Ca2+ response and AA release by stimulation of G protein-coupled receptors, we established several lines of Chinese hamster ovary cells expressing platelet-activating factor receptor or leukotriene B-4 receptor. Measurement of intracellular Ca2+ concentration ([Ca2+](i)) demonstrated that cell lines capable of releasing AA. elicited a sustained [Ca2+](i) increase when stimulated by agonists. The prolonged [Ca2+](i) elevation is the result of Ca2+ entry, because this elevation was blocked by EGTA treatment or in the presence of Ca2+ channel blockers (SKF 96365 and methoxyverapamil). cPLA(2) fused with a green fluorescent protein (cPLA(2)-GFP) translocated from the cytosol to the perinuclear region in response to increases in [Ca2+](i). When EGTA was added shortly after [Ca2+](i) increase, the cPLA(2)-GFP returned to the cytosol, without liberating AA. After a prolonged [Ca2+](i) increase, even by EGTA treatment, the enzyme was not readily redistributed to the cytosol. Thus, we propose that a critical time length of [Ca2+](i) elevation is required for continuous membrane localization and full activation of cPLA(2).