Abstract
The mechanism of inhibition of phosphatidylcholine biosynthesis by okadaic acid was investigated in suspension cultures of isolated rat hepatocytes. Cells were pulsed with [methyl-3H]choline and chased in the absence or presence of 1 microM 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 32P(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.
Highlights
Okadaic acid is a complex polyketal produced by marine dinoflagellates and is a potent andspecific inhibitor of protein phosphatases 1 and 2A [4].This compound is recognized as an excellent probe for studying cellular processes that are regulated by phosphorylation [5].We have demonstrated that in the phosphorylation of several peptides in okadaic incubation of the 10,000 X g supernatant from a rat liver acid-treated hepatocytes compared with controls. homogenate with okadaic acid inhibited the time-dependent
In thepresent study we demonstrate that thedecrease in PC biosynthesis observed in hepatocytes incubated with okadaic acid coincides directly with an increased phosphorylation of cytosolic CT
Biosynthesis of labeled PC increased with time incontrolincubations (Fig. 1).In the presence of okadaic acid, PC biosynthesis was notinhibited between 0 and 15 min, but was inhibited by 24% (p < 0.05) between 15 and 30 min and 50% (p< 0.05) between 30 and 60 min of incubation
Summary
Of Canada and thAelberta HeritageFoundation for Medical Research. Recipient of a graduateassistantship award from the Department 32Pwi ere obtained from Amersham Corp. Hepatocytes were isolated and incubated with [methyl-3H]choline(5 pCi/flask) for 30 min and subsequently chased in the absence or presence of 1PM okadaic acid for up to 120 min. For measurement of cellular DG, hepatocytes were incubated in the absence or presence of 1PM okadaic acid for up to 120 min, lipids were extracted as described [7], and the DG content in the lipid extract was determined [11].For measurement of microsomal DG, the hepatocytes were homogenized in 1 mlof 50 mM Tris-HC1, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 0.025% sodium azide, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride (buffer A). The hepatocytes were incubated with ["PIP, (200 pgCi/flask) for 60 min in Pi-free medium and subsequently incubated for 60 min with unlabeled Pi in the absence or presence of 1 P M okadaic acid. Student'st test wasused for the determination of statistical significance.The level of significancewas defined a s p < 0.05
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