LEUKOTRIENE B-4 STIMULATES THE RELEASE OF ARACHIDONATE IN HUMAN NEUTROPHILS VIA THE ACTION OF CYTOSOLIC PHOSPHOLIPASE A(2)

Leukotriene B-4 (LTB4) is a potent lipid mediator of inflammation and is involved in the receptor-mediated activation of a number of leukocy te responses including degranulation, superoxide formation, and chemot axis. Tn the present research, stimulation of unprimed polymorphonucle ar leukocytes (neutrophils) with LT4, results in the transient release of arachidonate as measured by mass. This release of arachidonate was maximal at an LTB4 concentration of 50-75 nM and peaked at 45 s after stimulation with LTB4. The transient nature of this release can be at tributed, in part, to a fast (< 60 s) metabolism of the added LTB4. Mo reover, 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 LTB4, which would be expected to more closely resemble th e situation in vivo where the cell may come into contact with an envir onment where LTB4 is in near constant supply, yielded a more sustained release of arachidonate. No release of [H-3]arachidonate was observed when using [H-3]arachidonate-labeled cells. This indicates that the r elease of arachidonate as measured by mass is most probably the result of hydrolysis of arachidonate-containing phosphatidylethanolamine wit hin the cell since the radiolabeled arachidonate is almost exclusively incorporated into phosphatidylcholine and phosphatidylinositol pools under the non-equilibrium radiolabeling conditions used. Consistent wi th the role of cytosolic phospholipase A(2) (cPLA(2)) in the release o f arachidonate, potent inhibition of the LTB4-stimulated release was o bserved with methylarachidonylfluorophosphonate, an inhibitor of cPLA( 2) (IC50 of 1 mu M). The bromoenol lactone of the calcium-independent phosphospholipase A(2). failed to affect LTB4-stimulated release of ar acbidonate in these cells. (C) 1997 Elsevier Science B.V.