Phosphatidic acid elicits calcium mobilization and actin polymerization through a tyrosine kinase-dependent process in human neutrophils: a mechanism for induction of chemotaxis

Abstract Phospholipids mediate important effects as extracellular messengers in diverse biological systems. We investigated the effects of phosphatidic acid, a biologically active phospholipid potentially involved in the inflammatory process, on calcium mobilization and actin polymerization in human neutrophils and correlated these effects with induction of chemotactic migration. Intermediate-chain length phosphatidic acid (DiC10-PA) induced a biphasic increase in intracellular Ca2+ characterized by a rapid rise commencing immediately upon addition of stimulus followed by a secondary increase which, unlike the initial response, was eliminated by chelation of extracellular Ca2+. Neither of these responses were induced by C10-lysophosphatidic acid or diacylglycerol. The tyrosine kinase inhibitor herbimycin-A (5–10 μg/ml) completely blunted the initial but not the delayed response effected by DiC10-PA. Long-chain phosphatidic acid (DiC18:1) induced only an initial rapid increase in intracellular Ca2+ and this response was similarly markedly attenuated by herbimycin-A. Among several physiologically relevant phospholipids, only phosphatidic acid was able to induce Ca2+ mobilization; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol — used individually or in mixed liposomes — were without effect. Phosphatidic acid conferred calcium-mobilizing activity upon inactive liposome preparations and phosphatidic acid-enriched cellular plasma membranes possessed similar calcium-mobilizing activity. Both DiC10-PA and DiC18:1-PA induced actin polymerization in neutrophils at rates which mirrored the influence of each agent on Ca2+ mobilization. Herbimycin-A blunted the initial increase in actin polymerization effected by phosphatidic acid but had no effect on the delayed, EGTA-sensitive phase. DiC10-PA and DiC18:1-PA also induced neutrophil migration along a concentration gradient. Phospholipids that failed to induce a calcium transient, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol, likewise failed to induce either actin polymerization or chemotactic migration. Unlike chemotaxis induced by zymosan-activated human serum, phosphatidate-induced chemotaxis was strongly inhibited by pretreatment of cells with herbimycin-A. Consistent with these observations, phosphatidic acid induced the tyrosine phosphorylation of several proteins as early as 10 s after stimulation. Phosphorylation of two distinct proteins with approximate molecular sizes of 72 and 82 kDa was inhibited by levels of herbimycin A used to effectively inhibit calcium mobilization, actin polymerization and chemotaxis. Thus, in neutrophilic leukocytes, extracellular phosphatidic acid induces a unique tyrosine kinase-based signalling pathway that results in calcium mobilization and actin polymerization. These processes may promote directed cellular migration as a consequence of the interaction of phosphatidic acid with neutrophil plasma membranes.