Eicosanoid Synthesis in Resident Macrophages: Role of Protein Kinase C

Abstract

In this study we investigated the effects of different agents known as activators or inhibitors of the calcium and phospholipid-dependent protein kinase (PKC) on the eicosanoid production in resident mouse peritoneal macrophages and in the murine macrophage-like cell line HA38. PKC activators such as the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA) or the synthetic cell-permeable diacylglycerols 1,2-dioctanoylglycerol (DiC8) and 1-oleoyl-2-acetylglycerol (OAG) stimulated arachidonic acid (AA) release from prelabelled cells and also decreased AA incorporation into cellular phospholipids within minutes. This rapid enlargement in the amount of free AA available for eicosanoid synthesis resulted in a significantly enhanced cellular prostaglandin E2 (PGE2) production, whereas leukotriene C4 (LTC4) could not be detected under these experimental conditions. Increasing the intracellular calcium level by simultaneous addition of calcium ionophores together with PKC activators in suboptimal concentrations led to a synergistic production of PGE2 and LTC4. PKC inhibitors such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), sphingosine, tamoxifen, and staurosporine, which exert their inhibitory action on PKC by different mechanisms, totally abolished TPA effects, whereas changes induced by (DiC8) or OAG were only partially reversed. Aluminum fluoride (AlF4 −) as an activator of regulatory guanine nucleotide binding proteins (G-proteins) elevated the intracellular concentration of inositolphosphates (IP) and diacylglycerols (DAG), thus leading to an activation of PKC and an increase in the intracellular calcium level, and subsequent enhanced eicosanoid production. AlF4 −-mediated PGEz and LTC4 synthesis again was reversed by using PKC inhibitors. Preincubation of the cells with pertussis toxin (PT) did not inhibit but even enhanced PGE2 production stimulated by zymosan or AlF4 −. Depletion of PKC activity by prolonged incubation of macrophages with TPA resulted in an extensive abrogation of the A1F4 − or zymosan-induced PGE2 secretion. These data provide further evidence that PKC is centrally involved in the regulation of macrophage eicosanoid synthesis initiated by different stimuli.