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 A2 (cPLA2) 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 B4 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). cPLA2 fused with a green fluorescent protein (cPLA2-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 cPLA2-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 cPLA2.
Hirabayashi
T
T
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Tokyo, Hongo, Toyko, Japan.
Kume
K
K
Hirose
K
K
Yokomizo
T
T
Iino
M
M
Itoh
H
H
Shimizu
T
T
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology*; D017931:DNA Primers; D004789:Enzyme Activation; D006168:Guinea Pigs; D006801:Humans; D017136:Ion Transport; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010980:Platelet Membrane Glycoproteins / Q000235:genetics; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D018102:Receptors, Leukotriene B4 / Q000235:genetics