The role of polyphosphoinositides and their breakdown products in A23187-induced release of arachidonic acid from rabbit polymorphonuclear leucocytes

Abstract

Stimulation of rabbit polymorphonuclear leucocytes with A23187 causes phospholipase C mediated breakdown of polyphosphoinositides, as evidenced by accumulation of [3H]inositol-labelled inositol bisphosphate and inositol trisphosphate. At the same time the polyphosphoinositides and the products oftheir breakdown, diacylglycerol and phosphatidic acid, label rapidly with radioactive arachidonic acid. Enhancement of polyphosphoinositide labelling is not as great as enhancement of diacylglycerol or phosphatidic acid labelling, suggesting additional early activation of a second independent synthetic pathway to the last named lipids. Experiments using double (3H/14C) labelling, to distinguish pools with different rates of turnover, suggest the major pool of arachidonic acid used for synthesis of lipoxygenase metabolites turns over more slowly than arachidonic acid in diacylglycerol, but at about the same rate as arachidonic acid esterified in phosphatidylcholine or phosphatidylinositol. Further, when cells are prelabelled with [14C]arachidonic acid, then stimulated for 5 min, it is only from phosphatidylcholine, and to a lesser extent phosphatidylinositol, that radiolabel is lost. Release of arachidonic acid is probably via phospholipase A2, since it is blocked by the phospholipase A2 inhibitor manoalide. The absence of accumulated lysophosphatides can be explained by reacylation and, in the case of lysophosphatidylinositol, deacylation. The importance of phospholipase A2 in phosphatidylinositol breakdown contrasts with the major role of phospholipase C in polyphosphoinositide hydrolysis. Measurements of absolute free fatty acid levels, as well as studies showing a correlation between production of radiolabelled hydroxyeicosatetraenoic acids and release of radiolabel from the phospholipid pool, both suggest that hydrolysis of arachidonic acid esterified into phospholipids is the limiting factor regulating formation of lipoxygenase metabolites. By contrast with A23187, fMet-Leu-Phe (a widely used polymorphonuclear leucocyte activator) is a poor stimulant for arachidonic acid release unless a 'second signal' (e.g. cytochalasin B, or a product of A23187-stimulated cells) is also present. In the presence of cytochalasin B, fMet-Leu-Phe, like A23187, stimulates release of radiolabelled arachidonic acid principally from phosphatidylcholine.

Selective incorporation of (15S)-hydroxyeicosatetraenoic acid in phosphatidylinositol of human neutrophils: agonist-induced deacylation and transformation of stored hydroxyeicosanoids.

“…Although evidence for agonist-induced release and further oxygenation of stored HETEs has not been previously reported with human PMNs, it is well established that incorporated AA can be released and transformed by PMNs exposed to appropriate stimuli (10,19,34). FMLP, PMA, and A23187 each led to the release of label from PI ( Fig.…”

Complement membrane attack on nucleated cells: resistance, recovery and non-lethal effects

“…The calcium ionophore A23187 stimulates via activation of phospholipase A2 (Meade et al, 1986) and MAC-induced phospholipase activation has recently been demonstrated in glomerular epithelial cells and shown to be calciumdependent (Cybulsky et al, 1988b). Removal of extracellular Ca2' abolishes MAC-induced secretion of arachidonic acid metabolites in neutrophils and monocytes, suggesting a similar mechanism Hansch et al, 1987).…”

“…Thereafter, arachidonic acid may be metabolized along one of two pathways, the cyclo-oxygenase pathway, widely distributed in mammalian cells and producing prostaglandins or thromboxanes, or the 5-lipoxygenase pathway, restricted to neutrophils, eosonophils, macrophages, monocytes and mast cells, and producing leukotrienes and HETEs (Salmon & Higgs, 1987). A role for calcium in control of these pathways is suggested by the observations that stimulation of the arachidonic acid cascade in macrophages by many stimuli is dependent on the presence of extracellular calcium and is inhibited by calcium antagonists like quinine (Aderem et al, 1986), and that the calcium ionophore A23187 stimulates arachidonic acid release in neutrophils (Meade et al, 1986). Calcium may be involved at several stages.…”

Ca2+ Ionophore and Phorbol Ester Stimulate Diacylglycerol Formation and Phosphatidylcholine Hydrolysis in Rat Parotid Acinar Cells

“…DAG formation in part through a protein kinase C in rat parotid acinar cells (13). In many cell types, Ca 2+ ionophores and phorbol esters are now known to in crease DAG levels through PC, but not phosphoinosi tide, hydrolysis (12,15,(19)(20)(21). In this study, we showed that both A23187 and PDBu stimulated DAG generation ( Figs.…”