Bradykinin Activates a Phospholipase D that Hydrolyzes Phosphatidylcholine in PC12 Cells

In PC12 pheochromocytoma cells whose phospholipids had been prelabeled with [ 3 H]palmitic acid, bradykinin increased the production of [ 3 H]phosphatidic acid. The increase in [ 3 H]phosphatidic acid occurred within 1–2 min, before the majority of the increase in [ 3 H]diacylglycerol. When the phospholipids were prelabeled with [ 3 H]choline, bradykinin increased the intracellular release of [ 3 H]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 [ 3 H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [ 3 H]phosphatidylethanol and [ 3 H]phosphatidylpropanol, respectively. The effect of bradykinin on [ 3 H]phosphatidic acid and [ 3 H]phosphatidylethanol formation was partially dependent on extracellular Ca 2+ . In cells treated with nerve growth factor, carbachol also increased [ 3 H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [ 14 C]stearic acid and [ 3 H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14 C/ 3 H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14 C/ 3 H ratio of phosphatidylcholine. The 14 C/ 3 H 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.