Leukotriene B 4 stimulates the release of arachidonate in human neutrophils via the action of cytosolic phospholipase A 2

Abstract

Leukotriene B 4 (LTB 4) is a potent lipid mediator of inflammation and is involved in the receptor-mediated activation of a number of leukocyte responses including degranulation, superoxide formation, and chemotaxis. In the present research, stimulation of unprimed polymorphonuclear leukocytes (neutrophils) with LTB 4 results in the transient release of arachidonate as measured by mass. This release of arachidonate was maximal at an LTB 4 concentration of 50–75 nM and peaked at 45 s after stimulation with LTB 4. The transient nature of this release can be attributed, in part, to a fast (<60 s) metabolism of the added LTB 4. Moreover, the inhibition of the reacylation of the released arachidonate with thimerosal results in greater than 4-times as much arachidonate released. Thus, a rapid reacylation of the released arachidonate also contributes to the transient nature of its measured release. Multiple additions of LTB 4, which would be expected to more closely resemble the situation in vivo where the cell may come into contact with an environment where LTB 4 is in near constant supply, yielded a more sustained release of arachidonate. No release of [ 3H]arachidonate was observed when using [ 3H]arachidonate-labeled cells. This indicates that the release of arachidonate as measured by mass is most probably the result of hydrolysis of arachidonate-containing phosphatidylethanolamine within the cell since the radiolabeled arachidonate is almost exclusively incorporated into phosphatidylcholine and phosphatidylinositol pools under the non-equilibrium radiolabeling conditions used. Consistent with the role of cytosolic phospholipase A 2 (cPLA 2) in the release of arachidonate, potent inhibition of the LTB 4-stimulated release was observed with methylarachidonylfluorophosphonate, an inhibitor of cPLA 2 (IC 50 of 1 μM). The bromoenol lactone of the calcium-independent phosphospholipase A 2. failed to affect LTB 4-stimulated release of arachidonate in these cells.