Influence of arachidonic acid on indices of phospholipase A2 activity in the human neutrophil

J. D. Winkler, C. M. Sung, W. C. Hubbard, F. H. Chilton

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Abstract

The present studies were conducted to understand better the regulation of phospholipase A2 (PLA2)-dependent mobilization of lipid mediators by arachidonic acid (C(20:4)). After stimulation of human neutrophils, g.l.c./m.s. analysis of non-esterified fatty acids indicated that the quantity of C(20:4) increased as a function of time after stimulation, from undetectable quantities to >800 pmol/107 cells. In contrast with C(20:4), the quantities of other free fatty acids such as oleic and linoleic were high in resting cells and did not change after stimulation. Some 15% of the C(20:4) released from cellular lipids remained cell-associated. To examine the effect of C(20:4) on its own release, neutrophils were exposed to [2H8]C(20:4), to differentiate it by g.l.c./m.s. from naturally occurring C(20:4). In A23187-stimulated neutrophils, low concentrations (5-10 μM) of [2H8]C(20:4) added just before A23187 increased the quantity of C(20:4) produced by the cell, whereas higher concentrations (30-50 μM) decreased the quantity of C(20:4) released from phospholipids. As other measures of PLA2 activity, the effects of C(20:4) on production of platelet-activity factor (PAF) and leukotriene B4 (LTB4) were assessed. C(20:4) treatment just before stimulation of neutrophils blocked PAF and LTB4 production in a concentration-dependent manner (IC50 10-20 μM). The effect of C(20:4) was not blocked by the cyclo-oxygenase inhibitor naproxine (10 μM), nor could it be mimicked by 1 μM LTB4, 5-hydroxyeicosa-6,8,11,14-tetraenoic acid (5HETE), 5-hydroperoxyeicosa-6,8,11,14-tetraenoic acid (5HPETE) or 15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15HETE). The 5-lipoxygenase (5LO) inhibitor zileuton induced a concentration-dependent decrease in PAF, with a maximal effect of a 50% decrease at 10-50 μM. The decrease in PAF by the 5LO inhibitor could not be circumvented by addition of 1 μM 5HETE, 5HPETE and LTB4, and may be attributed to the capacity of zileuton to increase the quantity of C(20:4) in A23187-treated neutrophils. The inhibitory effect of C(20:4) (20-40 μM) on PAF production could be antagonized by the protein kinase C inhibitor staurosporine (30 nM), but not by inhibitors of protein kinase A, tyrosine kinase or calmodulin kinase II. Taken together these data demonstrate that C(20:4) is selectively released from membrane phospholipids of A23187-stimulated neutrophils, and this C(20:4) may play an important role in regulating the mobilization of C(20:4) by altering PLA2 activity.

Original languageEnglish (US)
Pages (from-to)825-831
Number of pages7
JournalBiochemical Journal
Volume291
Issue number3
DOIs
StatePublished - 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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