INFLUENCE OF ARACHIDONIC-ACID ON INDEXES OF PHOSPHOLIPASE-A(2) 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 q uantity of C20:4 increased as a function of time after stimulation, fr om undetectable quantities to > 800 pmol/10(7) cells. In contrast with C20:4, the quantities of other free fatty acids such as oleic and lin oleic were high in resting cells and did not change after stimulation. Some 15% of the C20:4 released from cellular lipids remained cell-ass ociated. To examine the effect of C20:4 on its own release, neutrophil s were exposed to [H-2(8)C20:4, to differentiate it by g.l.c./m.s. fro m naturally occurring C20:4. In A23187-stimulated neutrophils, low con centrations (5-10 muM) of [H-2(8)C20:4 added just before A23187 increa sed the quantity of C20:4 produced by the cell, whereas higher concent rations (30-50 muM) decreased the quantity of C20:4 released from phos pholipids. 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 neutrophil s blocked PAF and LTB4 production in a concentration-dependent manner (IC50 10-20 muM). The effect of C20:4 was not blocked by the cyclo-oxy genase inhibitor naproxine (10 muM), nor could it be mimicked by 1 muM LTB4, 5-hydroxyeicosa-6,8,11,14-tetraenoic acid (5HETE), 5-hydroperox yeicosa-6,8,11,14-tetraenoic acid (5HPETE) or 15-hydroxyeicosa-5,8,11, 13-tetraenoic acid (15HETE). The 5-lipoxygenase (5LO) inhibitor zileut on induced a concentration-dependent decrease in PAF, with a maximal e ffect of a 50% decrease at 10-50 muM. The decrease in PAF by the 5LO i nhibitor could not be circumvented by addition of 1 muM 5HETE, 5HPETE and LTB4, and may be attributed to the capacity of zileuton to increas e the quantity of C20:4 in A23187-treated neutrophils. The inhibitory effect of C20:4 (20-40 muM) on PAF production could be antagonized by the protein kinase C inhibitor staurosporine (30 nM), but not by inhib itors of protein kinase A, tyrosine kinase or calmodulin kinase II. Ta ken together, these data demonstrate that C20:4 is selectively release d from membrane phospholipids of A23187-stimulated neutrophils, and th is C20:4 may play an important role in regulating the mobilization of C20:4 by altering PLA2 activity.