Synthesis of Sphingomyelin in Rat Lenses and in Canine Lens Epithelial Cells

Abstract

Abstract: : Purpose: This study of P-lipid metabolism examines the turnover rates of two essential membrane components, sphingomyelin (SM) and phosphatidylcholine (PtdC), in intact rat lenses and in dog lens epithelial cells (DLEC). SM and PtdC metabolism are related because choline enters the PtdC pool through the CDP-choline pathway, then SM is formed by transfer of the phosphocholine group from PtdC to ceramide. Methods: Intact lenses from 100 to 120 g rats were incubated in TC-199 medium. DLEC cultures were grown from canine capsule-epithelial explants in Dulbecco’s MEM + 20 % FBS. Lenses or third-passage DLEC in multi-well cell culture plates (9 wells/group) were incubated with [₃H]choline, 1µCi/ml. After 2 days, some groups of lenses or cells were «chased» in ₃H-free medium for up to 7 days. PtdC and SM were extracted, separated by TLC, and analyzed for ₃H. Data are expressed as mean (± SEM). Results: Intact rat lenses accumulated 8642 (± 722) dpm of [₃H]PtdC after 2 days with [₃H]choline, and 3.1 (± 0.4) % of this [₃H]PtdC was converted to [₃H]SM. After 5 more days with [₃H]choline, [₃H] increased 2.5-fold in PtdC and 3.4-fold in SM. PtdC label decreased by 23 (± 2) % after 5 days chase, while SM increased by 31 (± 4) %. Transfer of label from [₃H]PtdC to [₃H]SM was more rapid in DLEC. After 2 days with [₃H]choline, [₃H]SM was 9.6 ± (0.2) % of [₃H]P-lipids in recently-plated, rapidly-growing DLEC and was 5.4 (± 0.1) % of [₃H]P-lipids in newly-confluent DLEC. A 1-day chase resulted in loss of 65 (± 1.7) % of [₃H] from PtdC and 16 (± 2.9) % from SM in the confluent cells, and in a 4-day chase, 81 (± 1.1) % of PtdC and 30 (± 5.5) % of SM were lost. Disappearance of [₃H] from the P-lipids of the rapidly growing cells was significantly greater. Conclusion: Synthesis of SM from PtdC was more rapid in DLEC than in cultured, intact rat lenses, and a significant amount of [₃H] was lost from the P-lipids during a chase in unlabeled medium, indicating the presence of P-lipid degradation and turnover, even in rapidly growing cells.

Keywords: 458 lipids • 467 metabolism • 399 enzymes/enzyme inhibitors