Diacylglycerol generation in fluoride-treated neutrophils: Involvement of phospholipase D

Neutrophils exposed to fluoride ion (F^-) respond with a delayed and sustained burst of superoxide anion release that is both preceded by and dependent on the influx of Ca²⁺ from the extracellular medium. The results of this study demonstrate a similarly delayed and sustained generation of 1,2-diglyceride in F^--treated neutrophils, over 90% of which was 1,2-diacylglycerol. Diacylglycerol generation was not dependent on the presence of extracellular Ca²⁺. Conversely, in contrast to results obtained with other agonists, removal of extracellular Ca²⁺ markedly potentiated synthesis of diacylglycerol in F^--treated neutrophils. This effect was accompanied by a corresponding decrease in the recovery of phosphatidic acid. In either the presence or absence of extracellular Ca²⁺, phosphatidic acid accumulated before diacylglycerol in F^--treated cells, suggesting the latter was derived from the former. Consistent with this hypothesis, the phosphatidic acid phosphohydrolase inhibitor, propranolol, suppressed generation of diacylglycerol as it potentiated the accumulation of phosphatidic acid in F^--treated neutrophils. This effect was observed both in the presence and absence of extracellular Ca²⁺. Moreover, high levels of propranolol (160 μmol/L) effected complete inhibition of diacylglycerol generation in F^--treated neutrophils with a corresponding increase in phosphatidic acid generation. Phosphatidylethanol accumulated in neutrophils stimulated with F^- in the presence of ethanol. The extent of phosphatidylethanol accumulation at all time points after addition of F^- corresponded to decreased levels of both phosphatidic acid and diacylglycerol, indicating that phosphatidylethanol was derived from the phospholipase D-catalysed transphosphatidylation reaction. The results indicate that F^- activates a Ca²⁺-independent phospholipase D, which appears to be the major, if not sole, catalyst for both phosphatidic acid and diacylglycerol generation in F^--treated neutrophils. Ca²⁺, mobilized as a result of F^- stimulation and possibly as a consequence of phospholipase D activation, exerts a profound effect on cellular second messenger levels by modulating the conversion of phosphatidic acid to diacylglycerol.