Influence of arachidonic acid on indices of phospholipase A2 activity in the human neutrophil.
The present studies were conducted to understand better the regulation of phospholipase A2 (PLA2)-dependent mobilization of lipid mediators by arachidonic acid (C20:4). After stimulation of human neutrophils, g.l.c./m.s. analysis of non-esterified fatty acids indicated that the quantity of C20:4 increased as a function of time after stimulation, from undetectable quantities to > 800 pmol/10(7) cells. In contrast with C20:4, 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 C20:4 released from cellular lipids remained cell-associated. To examine the effect of C20:4 on its own release, neutrophils were exposed to [2H8]C20:4, to differentiate it by g.l.c./m.s. from naturally occurring C20:4. In A23187-stimulated neutrophils, low concentrations (5-10 microM) of [2H8]C20:4 added just before A23187 increased the quantity of C20:4 produced by the cell, whereas higher concentrations (30-50 microM) decreased the quantity of C20:4 released from phospholipids. As other measures of PLA2 activity, the effects of C20:4 on production of platelet-activity factor (PAF) and leukotriene B4 (LTB4) were assessed. C20:4 treatment just before stimulation of neutrophils blocked PAF and LTB4 production in a concentration-dependent manner (IC50 10-20 microM). The effect of C20:4 was not blocked by the cyclo-oxygenase inhibitor naproxine (10 microM), nor could it be mimicked by 1 microM 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 microM. The decrease in PAF by the 5LO inhibitor could not be circumvented by addition of 1 microM 5HETE, 5HPETE and LTB4, and may be attributed to the capacity of zileuton to increase the quantity of C20:4 in A23187-treated neutrophils. The inhibitory effect of C20:4 (20-40 microM) 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 C20:4 is selectively released from membrane phospholipids of A23187-stimulated neutrophils, and this C20:4 may play an important role in regulating the mobilization of C20:4 by altering PLA2 activity.
Division of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
J D
JD
Winkler
C M
CM
Sung
W C
WC
Hubbard
F H
FH
Chilton
D016718:Arachidonic Acid / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D005227:Fatty Acids / Q000097:blood; D005230:Fatty Acids, Nonesterified / Q000097:blood; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology; D007975:Leukotriene B4 / Q000096:biosynthesis / Q000494:pharmacology; D015289:Leukotrienes / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000097:blood*; D054467:Phospholipases A2; D010972:Platelet Activating Factor / Q000096:biosynthesis
D005227:Fatty Acids; D005230:Fatty Acids, Nonesterified; D006893:Hydroxyeicosatetraenoic Acids; D015289:Leukotrienes; D010972:Platelet Activating Factor; D007975:Leukotriene B4; D016718:Arachidonic Acid; D000001:Calcimycin; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; C025348:arachidonic acid 5-hydroperoxide; D010741:Phospholipases A; D054467:Phospholipases A2