Journal of Leukocyte Biology
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Originally published online as doi:10.1189/jlb.0807531 on January 24, 2008

Published online before print January 24, 2008
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(Journal of Leukocyte Biology. 2008;83:1019-1027.)
© 2008 by Society for Leukocyte Biology

The role of diacylglyceride generation by phospholipase D and phosphatidic acid phosphatase in the activation of 5-lipoxygenase in polymorphonuclear leukocytes

Dana Albert*,1, Carlo Pergola{dagger},1, Andreas Koeberle{dagger}, Gabriele Dodt{ddagger}, Dieter Steinhilber* and Oliver Werz{dagger},2

* Institute of Pharmaceutical Chemistry, University of Frankfurt, Frankfurt, Germany; and
{dagger} Department of Pharmaceutical Analytics, Institute of Pharmacy, and
{ddagger} Interfakultäres Institut für Biochemie, Eberhard-Karls-University Tuebingen, Tuebingen, Germany

2 Correspondence: Department of Pharmaceutical Analytics, Pharmaceutical Institute, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany. E-mail: oliver.werz{at}uni-tuebingen.de

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 Ca2+ 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 µM). 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 (EC50, ~1 µM) 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.

Key Words: leukotriene • arachidonic acid • inflammation • 1-oleoyl-2-acetyl-glycerol • ionophore






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