Stimulation of deacylation in Madin-Darby canine kidney cells. 12-O-tetradecanoyl-phorbol-13-acetate stimulates rapid phospholipid deacylation.

ABSTRACT

The tumor-promoting phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), stimulates Madin-Darby canine (MDCK) cells to deacylate cellular phospholipid and to produce prostaglandins. We have used this system to characterize the kinetics of deacylation of [3H]arachidonate and the further metabolism of arachidonate by the cyclooxygenase system. Stimulation of the appearance of [3H]arachidonic acid in extracellular fluids was found to be maximal 2 h after treatment with TPA and its subsequent removal. The production of prostaglandins then followed for up to 24 h. Phospholipase activity was not inhibited by indomethacin over the range of 0.01-100 micrograms/ml. In contrast, prostaglandin synthesis was inhibited at 1 microgram/ml indomethacin. Further, there was a significant stimulation of deacylation within 15 min in the presence of TPA that increased to nearly 30% of the total radioactivity within 1 h. Likewise, stimulation of prostaglandin production was detected within 15 min, but, unlike the deacylation process, did not increase significantly during TPA treatment. The source of arachidonic acid in the early stimulation period was found to be primarily phosphatidylethanolamine, but phosphatidylcholine and phosphatidylinositol were also deacylated. The results presented here argue that the phospholipase and cyclooxygenase are not tightly coupled in this system. Furthermore, we conclude that the earliest effect of TPA with regard to increased prostaglandin production in the MDCK cell is the direct stimulation of phospholipase activity.