Choline Cytidylyltransferase Activity and Phosphatidylcholine Synthesis in Confluent and Growing Porcine Lens Epithelial Cells

Abstract

Abstract: : Purpose: During cataractogenesis, the rate of synthesis of phosphatidylcholine (Ptd–Cho) and other phospholipids often increases relative to non–cataractous lenses. This may reflect membrane repair, however it is not known how Ptd–Cho synthesis is controlled in lens cells. In many tissues choline cytidylyltransferase (CCT) is the rate–limiting and controlling enzyme in Ptd–Cho biosynthesis. Preliminary studies of Ptd–Cho synthesis in galactosemic rat cataracts suggested that CCT may increase in these lenses, but the CCT activity in whole lenses was too low for more extensive studies. The current study examines the relationship between CCT activity and Ptd–Cho synthesis in growing or confluent porcine lens epithelial cells (PLEC). Methods: PLEC from 7–day–old piglets were plated at various densities into multi–well plates containing DMEM with 16% FBS. Cell growth and density were monitored by measuring the protein concentration. CCT activity was measured as incorporation of P–[¹⁴C]choline into CDP–choline and Ptd–Cho synthesis was measured by incubating cells with [³H]choline and extracting with chloroform:methanol (1:1). Results: Cell density (total protein/well) increased to a maximum within 2 – 3 weeks after transfer to the cell–culture plates. Ptd–Cho synthesis was most rapid during the period of cell growth, then leveled off to a baseline rate in the confluent cells. Changes in CCT activity (measured as either total activity or CCT/mg protein) followed a general pattern similar to changes in Ptd–Cho synthesis, i.e., CCT activity reached a maximum during cell growth and decreased after the cells became confluent. This suggests that changes in CCT activity may contribute to controlling the changes in Ptd–Cho synthesis. However, Ptd–Cho synthesis was not directly proportional to CCT activity, and the ratio of Ptd–Cho synthesis to CCT activity decreased as the cells became confluent, indicating that changes in CCT are not entirely responsible for controlling the changes in Ptd–Cho synthesis that occur PLEC cells. Conclusions: The results were consistent with CCT being the rate–limiting enzyme of Ptd–Cho synthesis in rapidly growing PLEC, but other control mechanisms must limit Ptd–Cho synthesis in confluent cells.

Keywords: lipids • metabolism • enzymes/enzyme inhibitors