5-Lipoxygenase products modulate the activity of the 85-kDa phospholipase A2 in human neutrophils.
Addition of submicromolar concentrations of arachidonic acid (AA) to human neutrophils induced a 2-fold increase in the activity of a cytosolic phospholipase A2 (PLA2) when measured using sonicated vesicles of 1-stearoyl-2-[14C]arachidonoylphosphatidylcholine as substrate. A similar increase in cytosolic PLA2 activity was induced by stimulation of neutrophils with leukotriene B4 (LTB4), 5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (5-HETE). LTB4 was the most potent of the agonists, showing maximal effect at 1 nM. Inhibition of 5-lipoxygenase with either eicosatetraynoic acid or zileuton prevented the AA-induced increase in PLA2 activity but had no effect on the response induced by LTB4. Furthermore, pretreatment of neutrophils with a LTB4-receptor antagonist, LY 255283, blocked the AA- and LTB4-induced activation of PLA2 but did not influence the action of 5-HETE. Treatment of neutrophils with pancreatic PLA2 also induced an increase in the activity of the cytosolic PLA2; this response was inhibited by both eicosatetraynoic acid or LY 255283. The increases in PLA2 activity in response to stimulation correlated with a shift in electrophoretic mobility of the 85-kDa PLA2, as determined by Western blot analysis, suggesting that phosphorylation of the 85-kDa PLA2 likely underlies its increase in catalytic activity. Although stimulation of neutrophils with individual lipoxygenase metabolites did not induce significant mobilization of endogenous AA, they greatly enhanced the N-formylmethionyl-leucyl-phenylalanine-induced mobilization of AA as determined by mass spectrometry analysis. Our findings support a positive-feedback model in which stimulus-induced release of AA or exocytosis of secretory PLA2 modulate the activity of the cytosolic 85-kDa PLA2 by initiating the formation of LTB4. The nascent LTB4 is then released to act on the LTB4 receptor and thereby promote further activation of the 85-kDa PLA2. Since 5-HETE and LTB4 are known to prime the synthesis of platelet-activating factor, the findings suggest that 85-kDa PLA2 plays a role in platelet-activating factor synthesis.
Wijkander
J
J
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157-1016, USA.
O'Flaherty
J T
JT
Nixon
A B
AB
Wykle
R L
RL
D015117:5,8,11,14-Eicosatetraynoic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D016718:Arachidonic Acid / Q000494:pharmacology; D001095:Arachidonic Acids / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D005089:Exocytosis; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology*; D007700:Kinetics; D007975:Leukotriene B4 / Q000494:pharmacology*; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D010741:Phospholipases A / Q000097:blood*; D054467:Phospholipases A2; D013777:Tetrazoles / Q000494:pharmacology; D017962:alpha-Linolenic Acid / Q000494:pharmacology
D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D013777:Tetrazoles; D017962:alpha-Linolenic Acid; D015117:5,8,11,14-Eicosatetraynoic Acid; C080828:5-oxo-6,8,11,14-eicosatetraenoic acid; D007975:Leukotriene B4; D016718:Arachidonic Acid; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; C029510:4,7,10,13-eicosatetraynoic acid; D001094:Arachidonate 5-Lipoxygenase; D010741:Phospholipases A; D054467:Phospholipases A2; C063823:LY 255283