ON THE MECHANISM OF THE OKADAIC ACID-INDUCED INHIBITION OF PHOSPHATIDYLCHOLINE BIOSYNTHESIS IN ISOLATED RAT HEPATOCYTES

The mechanism of inhibition of phosphatidylcholine biosynthesis by okadaic acid was investigated in suspension cultures of isolated rat hepatocytes. Cells were pulsed with [methyl-H-3]choline and chased in the absence or presence of 1-mu-M okadaic acid for up to 120 min. Phosphatidylcholine biosynthesis was inhibited after 15 min of chase. To see if okadaic acid altered the degree of phosphorylation of cytidylyltransferase (CT), hepatocytes were incubated with P-32(i) and chased in the absence or presence of okadaic acid. Okadaic acid caused a rapid (within 15 min) increase in the phosphorylation state of the cytosolic enzyme. Two-dimensional peptide map analysis revealed an increase in the phosphorylation of several peptides in okadaic acid-treated hepatocytes compared with controls. After 15 min of incubation of hepatocytes with okadaic acid, membrane CT activity was decreased and a corresponding increase in cytosolic CT activity was observed. In hepatocytes incubated with okadaic acid and oleate a correlation between membrane CT activity, diacylglycerol level, and phosphatidylcholine biosynthesis was observed. These data suggest that the concentration of diacylglycerol is responsible for the increase in membrane CT activity and subsequently phosphatidylcholine biosynthesis in oleate-treated cells. We postulate that the okadaic acid-induced decrease in phosphatidylcholine biosynthesis is due to an increase in the phosphorylation state of CT which promotes a translocation of CT activity from the membranes to the cytosol.