Bradykinin Activates a Phospholipase D that Hydrolyzes Phosphatidylcholine in PC12 Cells

In PC12 pheochromocytoma cells whose phospholipids had been prelabelled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1-2 min. before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin-stimulated cells: bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.