Phosphatidic acid binding to human neutrophils: effects on tyrosine kinase-regulated intracellular Ca2+ mobilization

Neutrophils provide an attractive model with which to characterize cellular effects of phosphatidic acid (PA) independently of effects triggered by lysophosphatidic acid (LPA), since these cells lack LPA receptors. We developed a novel method to quantitate binding of PA to neutrophils and neutrophil plasma membranes. Intact cells or subcellular fractions were immobilized on nitrocellulose membranes and incubated in a bath containing [32P]PA under various conditions, followed by rapid rinsing with a mild detergent (0.05% Tween 20) to minimize non-specific binding. With this method, dioctanoyl PA specifically ligated plasma-membrane binding sitesin a time- and temperature-dependent manner. Specific binding of (DiC8-PA was markedly potentiated by pre-treatment of cells or membranes with ecto-phosphatidic acid phosphohydrolase (PAPase) inhibitor dimethylsphingosine (DMS). Optimum binding of DiC8-PA to PAPase-inhibited cells occurred within 10 min at room temperature, increased linearly with the cell concentration used, and was not significantly affected by alteration of pH over the range of 5.5-8.5. Of several phosphatidic acid species examined, optimal specific binding to immobilized neutrophils was observed with DiC8-PA and dicapryl (DiC10) PA; dicaproyl (DiC6) PA bound weakly, whereas dimyristoyl (DiC14) PA and dipalmitoyl (DiC16) PA did not bind. Dioleoyl (DiC18:1) PA bound to immobilized cells, but this binding was essentially non-specific, in that it was not reduced by excess non-radioactive ligand. Various LPA preparations, including [32P] lyso-octanoyl (C8) PA and [32P] lyso-oleoyl (C18:1) PA, showed very low specific binding to neutrophils in this system. Specific binding of DiC8-PA and DiC10-PA preparations correlated well with the ability of each to effect the mobilization of intracellular Ca2+ in neutrophils. Ca2+ mobilization was characterized by two distinct phases; 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 results are consistent with the hypothesis that neutrophils have specific binding sites for phosphatidic acid, the occupation of which leads to rapid mobilization of intracellular free Ca2+ via activation of tyrosine kinases. The methods described in this report may facilitate the identification and characterization of functional phosphatidic acid receptors on neutrophil plasma membranes.