Tight connection between choline transport and phosphatidylcholine synthesis in MDCK cells

Abstract

In MDCK cells, choline uptake, the first step in the CDP-choline pathway for the biosynthesis of choline-containing phospholipids and osmolytes, occurs via both a transport system highly specific for choline and a non-specific pathway. The specific choline carrier is present at the apical domain of cells grown on dishes and is sodium-independent. Growing the cells on a permeant support results in the preferential localization of the specific choline carrier at the basolateral domain. To characterize the relationships between the choline uptake sites and the synthesis of phosphatidylcholine, MDCK cells were incubated with [Me-3H]choline and/or [Me-14C]choline for various times (up to 36 h) and the incorporation of label into phospholipids and water-soluble molecules was determined. For cells grown on dishes, addition of [Me-3H]choline at the apical side was followed by rapid incorporation of the label into the successive intermediates of the CDP-choline pathway. A comparable situation was found when growing the cells on a permeant support and adding the labelled choline at the basolateral side of the culture. On the other hand, radioactive choline added to the apical bath entered the CDP pathway to only a very low extent. Efflux experiments on cells loaded with choline from either the apical or the basolateral side demonstrate the existence of intracellular pools of choline. Addition of hemicholinium-3, an inhibitor of the specific choline carrier, markedly reduced the metabolism of choline taken up by the cells on the basolateral side but had no effect on that transported at the apical side. These results strongly suggest the existence of a tight connection between the entry of choline through the specific choline carrier and phosphatidylcholine synthesis in MDCK cells.