Distinct Arachidonate-releasing Functions of Mammalian Secreted Phospholipase A2s in Human Embryonic Kidney 293 and Rat Mastocytoma RBL-2H3 Cells through Heparan Sulfate Shuttling and External Plasma Membrane Mechanisms

We analyzed the ability of a diverse set of mammalian secreted phospholipase A2(sPLA2) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA2-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E2 production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA2-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA2-IIC allows it to release arachidonate. Heparin nonbinding sPLA2-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA2-V and -X, which are unique among sPLA2s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C4 and platelet-activating factor. sPLA2-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA2mutants and immunocytostaining and reveal that sPLA2-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells.