Involvement of cytosolic phospholipase A2 and secretory phospholipase A2 in arachidonic acid release from human neutrophils.
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.
Marshall
J
J
Division of Vascular Surgery, Max Bell Research Center, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada.
Krump
E
E
Lindsay
T
T
Downey
G
G
Ford
D A
DA
Zhu
P
P
Walker
P
P
Rubin
B
B
D016718:Arachidonic Acid / Q000378:metabolism*; D002118:Calcium / Q000502:physiology; D002469:Cell Separation; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism* / Q000502:physiology; D054467:Phospholipases A2; D013379:Substrate Specificity
D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium