12-O-TETRADECANOYLPHORBOL-13-ACETATE INHIBITS AMINOPHOSPHOLIPID TRANSLOCASE ACTIVITY AND MODIFIES THE LATERAL MOTIONS OF FLUORESCENT PHOSPHOLIPID ANALOGS IN THE PLASMA-MEMBRANE OF BOVINE AORTIC ENDOTHELIAL-CELLS

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a pot ent mitogenic factor which can replace the growth promoting activity o f basic fibroblast growth factor (bFGF) on bovine aortic endothelial c ells. However, TPA-treated cells lose their strict contact inhibition at confluence, which is a characteristic of cells grown in the presenc e of bFGF. We have examined whether these changes could be related to modifications of the transbilayer and lateral motions of fluorescent l ipids, namely -2-[6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] capro yl] - phosphatidylcholine (C6-NBD-PC), phosphatidylserine (C6-NBD-PS), and -phosphatidylethanolamine (CG-NBD-PE) inserted in the outer leafl et of the cell plasma membrane. In TPA-treated cells, the three fluore scent phospholipids remained located in the outer leaflet for at least 1 h at 20 degrees C after their insertion, indicating a blockade of t he aminophospholipid translocase activity which is normally present in the plasma membrane of bFGF-treated cells [1, 2]. TPA also induced a large increase in the percentage of CG NBD-PC and C6-NBD-PE probes whi ch were free to diffuse laterally. The mobile fractions nl reached val ues of similar to 100% for the two lipids, while for bFGF-treated cell s they were found around 85 and 75%, respectively. For the CG-NBD-PS p robe, M remained unchanged in bFGF and TPA-treated cells, at around 85 %. TPA treatment also induced a twofold increase in the lateral diffus ion coefficients of C6-NBD-PC and C6-NBD-PE, while that of CG-NBD-PS r emained nearly unchanged. These effects of TPA may be related to the o bserved loss of differentiated properties of vascular endothelial cell s and not to its mitogenic properties. (C) 1997 Academic Press.