Prolonged effects of cyclic AMP analogues of phosphatidylcholine biosynthesis in cultured rat hepatocytes

Abstract

The short- and long-term effects of cyclic AMP analogues and phosphodiesterase inhibitors on phosphatidylcholine biosynthesis in monolayer cultures of rat hepatocytes were investigated. All the compounds tested produced an inhibition of phosphatidylcholine biosynthesis for up to 6 h after addition to the hepatocyte medium. The reduced rate of lipid synthesis was a function of the concentration of cyclic AMP analogue and was independent of the concentration of choline in the medium. The proportion of choline oxidized to betaine was relatively unaffected. Choline was incorporated into hepatocytes by saturable and non-saturable mechanisms. Although the various cAMP analogues had different effects on choline uptake, chlorophenylthio-cAMP reduced uptake of choline by 28% for cells treated for 1.5–15 h. This analogue lowered the V max of the saturable component of choline transport by 3.6-fold. Prolonged incubation of the hepatocytes with cAMP analogues resulted in a reversal of the inhibition of phosphatidylcholine synthesis. After 15 h all the compounds tested stimulated the relative incorporation of [ methyl- 3H]choline into phosphatidylcholine. For hepatocytes incubated with chlorophenylthio-cAMP for 14–16 h, there was a 2.8-fold stimulation of the rate of phosphatidylcholine synthesis. The enzymes responsible for the conversion of choline into phosphatidylcholine were examined at various times after addition of the chlorophenylthio-cAMP to the hepatocyte medium. The reduced synthesis of phosphatidylcholine strongly correlated with inhibition of CTP:phosphocholine cytidylyltransferase activity. After 12 h of treatment with the analogue, the relative inhibition of the cytidylyltransferase activity was reversed.