OVEREXPRESSION OF RAT-LIVER CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE ACCELERATES PHOSPHATIDYLCHOLINE SYNTHESIS AND DEGRADATION

Two rat liver cDNAs encoding CTP:phosphocholine cytidylyltransferase ( CT-1 and CT2) were expressed in COS cells. The specific activity of CT in the microsomes increased approximately 20- or 100-fold after trans fection with CT-1 or CT-2, respectively, but there was only a 3-5-fold increase in the rate of [H-3]choline or [H-3]glycerol incorporation i nto phosphatidylcholine (PC). The phosphocholine pool decreased simila r to 40% in keeping with a stimulation of the CT-catalyzed reaction. T he CDP-choline pool increased 12-fold suggesting that the conversion o f CDP choline to PC, catalyzed by cholinephosphotransferase, could not keep pace with the CT-catalyzed reaction. This could account for the discrepancy between the increases in the amount of active (membrane-bo und) CT and the rate of PC synthesis. Incubation of CT-transfected cel ls with sodium oleate to increase the supply of cellular diacylglycero l resulted in a further 2-fold increase in the rate of PC synthesis. T his suggests that the diacylglycerol supply may be a Limiting factor i n the degree of stimulation of PC synthesis in CT-transfected COS cell s. Despite the increased rate of PC synthesis, the total cellular PC m ass increased only 17%, due to a 3-fold acceleration of the PC degrada tion rate. To determine which degradative pathway far PC was accelerat ed in the CT-transfected cells, we measured the pool sizes of several catabolites. Neither diacylglycerol nor phosphatidic acid mass was alt ered. The pool of glycerophosphocholine (GPC) was increased similar to 4-fold, and there was elevated release of GPC from the CT-transfected cells. The turnover of choline in GPC and lyse-PC was very slow compa red with that of choline, phosphocholine, or CDP-choline, suggesting t hat GPC and lyso-PC were derived from slowly degraded choline-labeled PC. The metabolism of GPC and lyse-PC was stimulated in the cells over expressing CT. These data suggest that PC synthesis and degradation ar e coordinated and that PC catabolism involving PC --> lyso-PC --> GPC is accelerated in COS cells overexpressing CT.