BRADYKININ ACTIVATES A PHOSPHOLIPASE-D THAT HYDROLYZES PHOSPHATIDYLCHOLINE IN PC12 CELLS

In PC12 pheochromocytoma cells whose phospholipids had been prelabeled with [H-3]palmitic acid, bradykinin increased the production of [H-3]phosphatidic acid. The increase in [H-3]phosphatidic acid occurred within 1-2 min, before the majority of the increase in [H-3]diacylglycerol. When the phospholipids were prelabeled with [H-3]choline, bradykinin increased the intracellular release of [H-3]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 [H-3]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [H-3]phosphatidylethanol and [H-3]phosphatidylpropanol, respectively. The effect of bradykinin on [H-3]phosphatidic acid and [H-3]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [H-3]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [C-14]stearic acid and [H-3]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The C-14/H-3 ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the C-14/H-3 ratio of phosphatidylcholine. The C-14/H-3 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.