Stimulated Nonspecific Transport of Phospholipids Results in Elevated External Appearance of Phosphatidylserine in ras-Transformed Fibroblasts

• Published in: Archives of biochemistry and biophysics 2000-09, Vol.381 (2), p.295-301
• Main Authors: Momchilova, Albena, Ivanova, Lidia, Markovska, Tania, Pankov, Roumen
• Format: Article
• Language: English
• Subjects: 3T3 Cells; aminophospholipid translocase; Animals; Biological Transport, Active; Calcium - metabolism; Carrier Proteins - metabolism; Cell Membrane - metabolism; Fibroblasts - metabolism; Genes, ras; lipid scramblase; membrane asymmetry; Membrane Lipids - metabolism; Membrane Proteins - metabolism; Mice; phosphatidylserine; Phosphatidylserines - metabolism; Phospholipid Transfer Proteins; Phospholipids - metabolism; ras transformation; Transformation, Genetic
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.2000.1986

The content of phosphatidylserine (PS) was found to be increased three times in the plasma membrane outer leaflet of ras-transformed fibroblasts compared to their nontransformed counterparts. In an attempt to determine the mechanisms responsible for the enhanced external appearance of PS, we investigated the activities of aminophospholipid translocase and the nonspecific lipid scramblase. Both transport systems could separately or in combination contribute to PS accumulation in the extracellular leaflet. Aminophospholipid transfer was assessed by measuring the rate of NBD-PS internalization, and scramblase activity was estimated from the internalization of NBD-PC. The results showed that the aminophospholipid transport was inhibited and the nonspecific transport was stimulated in ras-transformed cells. To assess which of these two transport systems was related to elevation of PS external appearance, each of them was submitted to reversible alterations and the content of PS was measured simultaneously. Aminophospholipid translocase activity was inhibited by pyridyldithioethylamine treatment and reversed by reduction with dithiothreitol. Scramblase activity was modulated by a calcium repletion–depletion procedure. Calcium depletion was performed by cell incubation with BAPTA-AM and EGTA as Ca2+ intracellular and extracellular chelators. Restoration of the intracellular Ca2+ was achieved by cell incubation with Ca2+ and Ca2+-ionophore A23187. The results showed that the changes in PS outer appearance did not correlate with the uptake of NBD-PS but were closely related to NBD-PC internalization, suggesting that the nonspecific bidirectional lipid transfer was the major transport system translocating PS to the outer leaflet in ras-transformed cells.