PHOSPHATIDIC-ACID BINDING TO HUMAN NEUTROPHILS - EFFECTS ON TYROSINE KINASE-REGULATED INTRACELLULAR CA2+ MOBILIZATION

Neutrophils provide an attractive model with which to characterize cel lular effects of phosphatidic acid (PA) independently of effects trigg ered by lysophosphatidic acid (LPA), since these cells lack LPA recept ors. We developed a novel method to quantitate binding of PA to neutro phils and neutrophil plasma membranes. Intact cells or subcellular fra ctions were immobilized on nitrocellulose membranes and incubated in a hath containing [P-32]PA under various conditions, followed by rapid rinsing with a mild detergent (0.05% Tween 20) to minimize non-specifi c binding. With this method, dioctanoyl PA (DiC8-PA) specifically liga ted plasmamembrane binding sites in a time- and temperature-dependent manner. Specific binding of DiC8-PA was markedly potentiated by pre-tr eatment of cells or membranes with ecto-phosphatidic acid phosphohydro lase (PAPase) inhibitor dimethylsphingosine (DMS). Optimum binding of DiC8-PA to PAPase-inhibited cells occurred within 10 min at room tempe rature, increased linearly with the cell concentration used, and was n ot significantly affected by alteration of CH over the range of 5.5-8. 5. Of several phosphatidic acid species examined, optimal specific bin ding to immobilized neutrophils was observed with DiC8-PA and dicapryl (DiC10) PA; dicaproyl (DiC6) PA bound weakly, whereas dimyristoyl (Di C14) PA and dipalmitoyl (DiC16) PA did not bind. Dioleoyl (DiC18:1) PA bound to immobilised cells, but this binding was essentially non-spec ific, in chat it was not reduced by excess non-radioactive ligand. Var ious LPA preparations, including [P-32] lyso-octanoyl (C8) PA and [P-3 2] lyso-oleoyl (C18:1) PA, showed very low specific binding to neutrop hils in this system. Specific binding of DiC8-PA and DiC10-PA preparat ions correlated well with the ability of each to effect the mobilizati on of intracellular Ca2+ in neutrophils. Ca2+ mobilisation was charact erized by two distinct Chases; a rapid rise that was inhibited in the presence of the tyrosine kinase inhibitor herbimycin-A, followed by a sustained increase that was eliminated in the presence of EGTA. The re sults are consistent with the hypothesis that neutrophils have specifi c binding sites for phosphatidic acid, the occupation of which leads t o rapid mobilization of intracellular free Ca2+ via activation of tyro sine kinases. The methods described in this report may facilitate the identification and characterization of functional phosphatidic acid re ceptors on neutrophil plasma membranes.