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Involvement of Cytosolic Phospholipase A₂ and Secretory Phospholipase A₂ in Arachidonic Acid Release from Human Neutrophils¹

John Marshall^*, Eric Krump^2,, Thomas Lindsay^*, Gregory Downey, David A. Ford^§, Peihong Zhu^*, Paul Walker^* and Barry Rubin^3,*

Divisions of ^* Vascular Surgery and Respiratory Diseases, Max Bell Research Center, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada; Division of Cell Biology, Sick Children’s Hospital, Toronto, Ontario, Canada; and ^§ Department of Biochemistry and Molecular Biology, St. Louis University Health Sciences Center, St. Louis, MO 63104

The purpose of this study was to define the role of secretory phospholipase A₂ (sPLA₂), calcium-independent PLA₂, and cytosolic PLA₂ (cPLA₂) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils. While fMLP induced the release of extracellular sPLA₂ activity and AA, 70% of sPLA₂ activity remained associated with the cell. Treatment with the cell-impermeable sPLA₂ inhibitors DTT or LY311-727, or the anti-sPLA₂ Ab 3F10 all inactivated extracellular sPLA₂ activity, but had minimal effect on neutrophil AA mass release. In contrast, coincubation of streptolysin-O toxin-permeabilized neutrophils with DTT, LY311-727, or 3F10 all decreased [³H₈]AA release from [³H₈]AA-labeled, fMLP-stimulated cells. Exposure to fMLP resulted in a decrease in the electrophoretic mobility of cPLA₂, a finding consistent with cPLA₂ phosphorylation, and stimulated the translocation of cPLA₂ from cytosolic to microsomal and nuclear compartments. The role of cPLA₂ was further evaluated with the cPLA₂ inhibitor methyl arachidonyl fluorophosphonate, which attenuated cPLA₂ activity in vitro and decreased fMLP-stimulated AA mass release by intact neutrophils, but had no effect on neutrophil sPLA₂ activity. Inhibition of calcium-independent PLA₂ with haloenol lactone suicide substrate had no effect on neutrophil cPLA₂ activity or AA mass release. These results indicate a role for cPLA₂ and an intracellular or cell-associated sPLA₂ in the release of AA from fMLP-stimulated human neutrophils.