Choline metabolism and phosphatidylcholine biosynthesis in cultured rat hepatocytes

Abstract

Adult rat hepatocytes were isolated by collagenase perfusion and were maintained in monolayer culture for 24 h. Choline metabolism and phosphatidylcholine [PC] biosynthesis were studied in these cells by performing pulse-chase studies at physiological concentrations (1-40 .mu.M) of (Me-3H)-labeled or unlabeled choline in the culture medium. During the 15 min pulse incubation, choline entering the cells was rapidly phosphorylated to phosphocholine or oxidized to betaine. Low concentrations of choline in the medium decreased the relative amount of choline oxidized. During the 3 h chase period, the radioactivity in the phosphocholine pool was transferred to PC. Very little radioactivity was associated with CDP-choline. The rate-limiting step for PC biosynthesis in these cultured hepatocytes is the conversion of phosphocholine into CDP-choline. Similar results were obtained for all concentrations of choline in the culture medium. Cellular concentrations of phosphocholine were unaffected by the concentration of choline (1-40 .mu.M) in the medium. The majority of the label associated with betaine was secreted into the culture medium during the chase incubation. From the pulse-chase studies and the cellular phosphocholine concentrations, it was possible to estimate the rate of PC biosynthesis (2.2, 2.8, 3.1 and 3.7 nmol/min per g wet weight of cells cultured in 1, 5, 10 and 40 .mu.M-choline, respectively, for up to 4.25 h).