SEPARATION OF AGONIST-STIMULATED ARACHIDONATE MOBILIZATION FROM SUBSEQUENT LEUKOTRIENE B-4 SYNTHESIS IN HUMAN NEUTROPHILS - DIFFERENT EFFECTS OF OLEOYLACETYLGLYCEROL AND PHORBOL-MYRISTATE ACETATE AS PRIMING AGENTS

Preincubation of human neutrophils with phorbol esters or soluble digl ycerides enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidon ate mobilization and leukotriene B-4 (LTB(4)) synthesis. We have recen tly reported that 1,3-dioctanoylglycerol (1,3-diC8) is equipotent with 1,2-sn-dioctanoylglycerol (1,2-diC8) as a priming agent, thus suggest ing that the priming effects of diacylglycerols are protein kinase C ( PKC) independent (Rosenthal et al., 1993, Biochim. Biophys. Acta 1177: 79-86). In order to further investigate this question, the present stu dy has directly compared the effects of oleoylacetylglycerol (OAG) and the PKC activator, phorbol '12-myristate 13-acetate (PMA), on agonist -stimulated lipid metabolism. The results indicate that both OAG and P MA dose dependently enhance f-Met-Leu-Phe (fMLP)-stimulated release of [H-3]arachidonate. Optimal concentrations of OAG (5 mu m) and PMA (10 nM) are equipotent in increasing fMLP-stimulated arachidonate mobiliz ation as quantitated either with total radioactivity or by mass measur ements of free arachidonate. By contrast OAG is sixfold more effective than PMA in enhancing synthesis of 5-lipoxygenase (5-LO) metabolites by mass and two to threefold more effective than PMA in enhancing synt hesis of [H-3]eicosanoids. Furthermore, OAG, but not PMA, enhances fML P-stimulated synthesis of platelet-activating factor. By contrast, PMA directly stimulates [H-3]arachidonate mobilization, while OAG (20 mu M) does not; despite these differences, the combined effects of PMA OAG on subsequent agonist-stimulated arachidonate release are not grea ter than those of PMA alone. In cells challenged with subthreshold con centrations (<0.1 mu M) Of the calcium ionophore A23187, both OAG and PMA stimulate [(3)Harachidonate release but not [H-3]LTB(4) synthesis. These findings suggest that OAG does not directly activate 5-LO, but instead couples arachidonate mobilization to leukotriene synthesis in a PKC-independent manner. (C) 1994 Wiley-Liss, Inc.