5-hydroxy-6-8-11-14-eicosatetraenoic-acid and 1-oleoyl-2-acetylglycerol

Preincubation of human neutrophils with phorbol esters or soluble diglycerides enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization and leukotriene B4 (LTB4) synthesis. We have recently reported that 1,3-dioctanoylglycerol (1,3-diC8) is equipotent with 1,2-sn-dioctanoylglycerol (1,2-diC8) as priming agent, thus suggesting 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 study 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 PMA dose dependently enhance f-Met-Leu-Phe (fMLP)-stimulated release of [3H]arachidonate. Optimal concentrations of OAG (5 microns) and PMA (10 nM) are equipotent in increasing fMLP-stimulated arachidonate mobilization as quantitated either with total radioactivity or by mass measurements 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 synthesis of [3H]eicosanoids. Furthermore, OAG, but not PMA, enhances fMLP-stimulated synthesis of platelet-activating factor. By contrast, PMA directly stimulates [3H]arachidonate mobilization, while OAG (20 microM) does not; despite these differences, the combined effects of PMA + OAG on subsequent agonist-stimulated arachidonate release are not greater than those of PMA alone. In cells challenged with subthreshold concentrations (< 0.1 microM) of the calcium ionophore A23187, both OAG and PMA stimulate [3H]arachidonate release but not [3H]LTB4 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.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Arachidonic Acid; Biological Transport; Calcimycin; Diglycerides; Eicosanoids; Fatty Acids; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Leukotriene Antagonists; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases A; Tetradecanoylphorbol Acetate

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Cytochalasin B; Diglycerides; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases; Phospholipases A; Phospholipases A2

Diglyceride activators of protein kinase C (i.e., 1-0-myristoyl-, 1-0-palmitoyl-, and 1-0-oleoyl-2-acetylglycerol) interacted synergistically with an arachidonate metabolite, 5-hydroxyicosatetraenoate, to stimulate neutrophil degranulation and superoxide anion generation. Contrastingly, combinations of 15-hydroxyicosatetraenoate with the glycerides or 5-hydroxyicosatetraenoate with a dialkylglyceride (1-0-hexadecyl-2-ethylglycerol) produced no such synergy. The data support a view of stimulus-response coupling wherein protein kinase C is activated in parallel with the mobilization of arachidonate. Respective products of these events, e.g., phosphorylated proteins and hydroxyicosatetraenates, then interact to mediate function.

Topics: Arachidonic Acid; Arachidonic Acids; Cytoplasmic Granules; Diglycerides; Drug Synergism; Enzyme Activation; Glucuronidase; Humans; Hydroxyeicosatetraenoic Acids; Muramidase; Neutrophils; Protein Kinase C; Protein Kinases; Superoxides