Abstract
The mechanism by which glucagon and cAMP analogues inhibit phosphatidylcholine biosynthesis was investigated in rat hepatocytes. The studies were facilitated by preparation of an antibody to a synthetic peptide (D-F-V-A-H-D-D-I-P-Y-S-S-A) corresponding to residues 164-176 of CTP:phosphocholine cytidylyl-transferase. The antibody, which was purified by affinity chromatography, quantitatively immunoprecipitated cytidylyltransferase from rat liver cytosol. Various analogues of cAMP had no effect on the labeling of cytidylyltransferase with 32Pi in rat hepatocytes. Nor did the cAMP analogues have any effect on the distribution of cytidylyltransferase between cytosol and membranes. These results indicate that the supply of CDP-choline does not limit phosphatidylcholine biosynthesis in hepatocytes treated with cAMP analogues. A decreased supply of diacylglycerol was considered as an alternative mechanism for inhibition of phosphatidylcholine biosynthesis. An approximately 30% decrease in diacylglycerol concentration was observed in hepatocytes treated with the cAMP analogues or glucagon, compared with controls. A similar decrease of phosphatidylcholine biosynthesis was observed. The cAMP-mediated decrease in diacylglycerol levels and inhibition of phosphatidylcholine biosynthesis were reversed by addition of 0.5-1.5 mM oleic acid to the treated hepatocytes. A correlation coefficient of 0.93 was calculated between the levels of diacylglycerol and the rate of phosphatidylcholine biosynthesis. In another approach, the diacylglycerol levels were increased by an inhibitor of diacylglycerol lipase (U-57908) which also reversed the cAMP effects on diacylglycerol levels and phosphatidylcholine biosynthesis. We conclude that the cAMP-mediated inhibition of phosphatidylcholine biosynthesis was not due to an effect on the phosphorylation of cytidylyltransferase. Instead, phosphatidylcholine biosynthesis appears to be inhibited due to a decreased level of diacylglycerol, a substrate for CDP-choline: 1,2-diacylglycerol cholinephosphotransferase.
Highlights
Evidence That Cyclic AMP-induced Inhibition of Phosphatidylcholine Biosynthesis Is Caused by a Decrease in Cellular Diacylglycerol Levels in Cultured Rat Hepatocytes*
Cyclic AMP analogues inhibit PC synthesis in cultured crease in diacylglycerol concentration was observed in hepatocytes in short term incubation(s11).The inhibitionof hepatocytes treated with the cAMP analogues or glu- PC biosynthesisis accompanied by partialinactivation of cagon, compared with controls
The [11].Okadaicacid, a potent protein phosphatase inhibitor, CAMP-mediated decrease in diacylglycerol levels and inhibits PC synthesis in rat hepatocytes and increases inhibition of phosphatidylcholinebiosynthesiswere re- cytosolic C T activity with a correspondingdecrease in activity versed by addition of 0.5-1.5 mM oleic acid to the on the membrane fraction[12]
Summary
Actiuity-Two different methods, digitonin permeabilization and homogenization and centrifugation, were used to prepare cytosolic and membrane samples from CAMP analogue-treated hepatocytes. Phospho[methyl-3H]choline was used as a substrate, and the reaction was stopped by immersion of the assay tubes in boiling water for 1 min. After 5 min, the’medium was removed and cells were harvested with 2.0 ml methanol/water (1:1) and sonicated. Lipids in the remaining methanol/water solution were extracted by the method of Bligh and Dyer [29]. MM CPTcAMP, the medium was recovered from two 60-mm dishes and the cells were harvested with 2.0 ml of methanol/water (1:l) and sonicated.Duplicate 50-~1aliquots were recoveredfor protein determination. Extraction of the remaining methanol/water solution was performed by the method of Bligh and Dyer [29]. The ethanol solution was evaporated to dryness under Nz, and the amount of choline was determined by quantitative conversion of choline to phosphocholine as described in [30].
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