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

Abstract

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent mitogenic factor which can replace the growth promoting activity of basic fibroblast growth factor (bFGF) on bovine aortic endothelial cells. However, TPA-treated cells lose their strict contact inhibition at confluence, which is a characteristic of cells grown in the presence of bFGF. We have examined whether these changes could be related to modifications of the transbilayer and lateral motions of fluorescent lipids, namely 1-acyl-2-[6-[N-(7-nitrobenz2-oxa-1,3-diazol-4-yl)amino]caproyl]-phosphatidyl-choline (C6-NBD-PC), -phosphatidylserine (C6-NBD-PS), and -phosphatidylethanolamine (C6-NBD-PE) inserted in the outer leaflet of the cell plasma membrane. In TPA-treated cells, the three fluorescent phospholipids remained located in the outer leaflet for at least 1 h at 20°C after their insertion, indicating a blockade of the 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 C6-NBD-PC and C6-NBD-PE probes which were free to diffuse laterally. The mobile fractionsMreached values of ∼100% for the two lipids, while for bFGF-treated cells they were found around 85 and 75%, respectively. For the C6-NBD-PS probe,Mremained unchanged in bFGF and TPA-treated cells, at around 85%. TPA treatment also induced a twofold increase in the lateral diffusion coefficients of C6-NBD-PC and C6-NBD-PE, while that of C6-NBD-PS remained nearly unchanged. These effects of TPA may be related to the observed loss of differentiated properties of vascular endothelial cells and not to its mitogenic properties.