The role of diacylglyceride generation by phospholipase D and phosphatidic acid phosphatase in the activation of 5-lipoxygenase in polymorphonuclear leukocytes.
Diacylglycerides (DAGs) such as 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulate 5-lipoxygenase (5-LO) enzyme activity and function as agonists for human polymorphonuclear leukocytes (PMNL) to induce 5-LO product synthesis. Here, we addressed the role of endogenous DAG generation in agonist-induced 5-LO activation in human PMNL. Preincubation of PMNL with the phospholipase D (PLD) inhibitor 1-butanol potently suppressed 5-LO product synthesis induced by the Ca(2)(+) ionophore A23187 or thapsigargin (TG) and blocked A23187-evoked translocation of 5-LO from the cytosol to the nuclear membrane, analyzed by subcellular fractionation as well as by indirect immunofluorescence microscopy. Tertiary-butanol, a rather poor inhibitor of PLD, caused only moderate suppression of 5-LO and hardly inhibited 5-LO translocation. Interestingly, 1-butanol failed to inhibit 5-LO product formation when PMNL were stimulated with OAG (30 microM). Moreover, coincubation of A23187- or TG-stimulated PMNL with OAG reversed inhibition of 5-LO product formation by 1-butanol in a concentration-dependent manner (EC(50), approximately 1 muM) and also restored 5-LO translocation. In addition, inhibition of phosphatidic acid phosphatase (PA-P) by propranolol or bromoenol lactone caused suppression of 5-LO product formation and of translocation, which could be reversed by addition of exogenous OAG. Together, our data suggest that in agonist-stimulated PMNL, the endogenous formation of DAGs via the PLD/PA-P pathway determines 5-LO activation.
Albert
Dana
D
Institute of Pharmaceutical Chemistry, University of Frankfurt, Frankfurt, Germany.
Pergola
Carlo
C
Koeberle
Andreas
A
Dodt
Gabriele
G
Steinhilber
Dieter
D
Werz
Oliver
O
D020001:1-Butanol / Q000494:pharmacology; D000328:Adult; D001094:Arachidonate 5-Lipoxygenase / Q000097:blood*; D000001:Calcimycin / Q000494:pharmacology; D004075:Diglycerides / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D016859:Lipoxygenase Inhibitors; D008856:Microscopy, Fluorescence; D009504:Neutrophils / Q000201:enzymology*; D010711:Phosphatidate Phosphatase / Q000097:blood*; D010739:Phospholipase D / Q000097:blood*; D012016:Reference Values; D019284:Thapsigargin / Q000494:pharmacology
D004075:Diglycerides; D016859:Lipoxygenase Inhibitors; D000001:Calcimycin; D019284:Thapsigargin; D020001:1-Butanol; D001094:Arachidonate 5-Lipoxygenase; D010711:Phosphatidate Phosphatase; D010739:Phospholipase D