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

J Leukoc Biol. 2008 Apr;83(4):1019-27. doi: 10.1189/jlb.0807531. Epub 2008 Jan 24.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Butanol / pharmacology
  • Adult
  • Arachidonate 5-Lipoxygenase / blood*
  • Calcimycin / pharmacology
  • Diglycerides / metabolism*
  • Enzyme Activation
  • Humans
  • Lipoxygenase Inhibitors
  • Microscopy, Fluorescence
  • Neutrophils / enzymology*
  • Phosphatidate Phosphatase / blood*
  • Phospholipase D / blood*
  • Reference Values
  • Thapsigargin / pharmacology


  • Diglycerides
  • Lipoxygenase Inhibitors
  • Calcimycin
  • Thapsigargin
  • 1-Butanol
  • Arachidonate 5-Lipoxygenase
  • Phosphatidate Phosphatase
  • Phospholipase D