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Published online before print January 24, 2008

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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0807531

Received for publication August 10, 2007.
Revised November 28, 2007.
Accepted for publication December 17, 2007.

Article

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

Dana Albert ^*, Carlo Pergola , Andreas Koeberle , Gabriele Dodt , Dieter Steinhilber ^*, and Oliver Werz ^@

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

^@ To whom correspondence should be addressed. E-mail: oliver.werz{at}uni-tuebingen.de.

	Abstract

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