5-Lipoxygenase Products Modulate the Activity of the 85-kDa Phospholipase A in Human Neutrophils

doi:10.1074/jbc.270.44.26543

5-Lipoxygenase Products Modulate the Activity of the 85-kDa Phospholipase A in Human Neutrophils (*)

From the Departments of Biochemistry and Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157-1016

^¶ To whom correspondence should be addressed: Tel.: 910-716-4372; Fax: 910-716-7671.

Present address: Dept. of Physiological Chemistry, Lund University, P. O. 94, S-221 00 Lund, Sweden.

Abstract

Addition of submicromolar concentrations of arachidonic acid (AA) to human neutrophils induced a 2-fold increase in the activity of a cytosolic phospholipase A (PLA) when measured using sonicated vesicles of 1-stearoyl-2-[C]arachidonoylphosphatidylcholine as substrate. A similar increase in cytosolic PLA activity was induced by stimulation of neutrophils with leukotriene B (LTB), 5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (5-HETE). LTB 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 PLA activity but had no effect on the response induced by LTB. Furthermore, pretreatment of neutrophils with a LTB-receptor antagonist, LY 255283, blocked the AA- and LTB-induced activation of PLA but did not influence the action of 5-HETE. Treatment of neutrophils with pancreatic PLA also induced an increase in the activity of the cytosolic PLA; this response was inhibited by both eicosatetraynoic acid or LY 255283.

The increases in PLA activity in response to stimulation correlated with a shift in electrophoretic mobility of the 85-kDa PLA, as determined by Western blot analysis, suggesting that phosphorylation of the 85-kDa PLA 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 PLA modulate the activity of the cytosolic 85-kDa PLA by initiating the formation of LTB. The nascent LTB is then released to act on the LTB receptor and thereby promote further activation of the 85-kDa PLA. Since 5-HETE and LTB are known to prime the synthesis of platelet-activating factor, the findings suggest that 85-kDa PLA plays a role in platelet-activating factor synthesis.

Footnotes

↵* This work was supported by National Institutes of Health Grants AI 17287, HL 26818, HL 26257, and P01-HL 50395. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

AA

arachidonic acid

PAF

platelet-activating factor

PLA

phospholipase A

LTB

leukotriene B

5-HETE

5-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid

PMN

polymorphonuclear neutrophil

BSA

bovine serum albumin

ETYA

eicosatetraynoic acid

5-oxoETE

5-oxo-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid

15-oxoETE

15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid

compound I

(5R,12R)-6,8,10,14-(Z,Z,Z,E)-eicosatetraenoic acid

fMLP

N-formylmethionyl-leucyl-phenylalanine

PBS

phosphate-buffered saline

LPS

lipopolysaccharide.
↵²J. Wijkander, J. T. O'Flaherty, and R. L. Wykle, unpublished observation.
- Received March 13, 1995.
- Revision received August 24, 1995.