Differences in the Transbilayer and Lateral Motions of Fluorescent Analogs of Phosphatidylcholine and Phosphatidylethanolamine in the Apical Plasma Membrane of Bovine Aortic Endothelial Cells

Abstract

In the plasma membrane of various eucaryotic cell types, in particular blood platelets and erythrocytes, it is known that phospholipids are asymmetrically distributed between the two leaflets of the lipid bilayer and that this transverse asymmetry is controlled by an aminophospholipid translocase activity. In this respect, it was of interest to check whether there are differential transbilayer movements between amino- and neutral phospholipids in the apical plasma membrane of vascular endothelial cells which form the inner nonthrombogenic lining of the large blood vessel. In the first step we compared the transbilayer localization and also the rate of lateral motion of two fluorescent analogs of phosphatidylcholine and phosphatidylethanolamine, namely C6-NBD-PC and C6-NBD-PE, inserted into the apical plasma membrane of bovine aortic endothelial cells, in vitro. By the use of back-exchange experiments we have found that C6-NBD-PC could be removed from the cell membrane toward the culture medium regardless of the incubation conditions used, i.e., just after cell labeling at 0°C or even after further cell incubation for 1 h at 0 or 20°C. In contrast, C6-NBD-PE could be removed only when the cells were maintained at 0°C. After incubation for 1 h at 20°C, 85% of the probe molecules remained nonexchangeable, indicating probe translocation from the outer to the inner leaflet of the lipid bilayer. This "flip" process, which occurred at 20°C, was abolished when the endothelial cells were preincubated with N-ethylmaleimide, diamide, vanadate (VO 3- 4) and vanadyl (VO 2+) ions, a set of substances which inhibit aminophospholipid translocase activity in various systems, and with a combination of sodium azide and 2-deoxyglucose which led to nearly complete ATP depletion in the cells. Fluorescence recovery after photobleaching experiments were also carried out to specify more precisely the localization and dynamics of the probes in the two leaflets of the plasma membrane lipid bilayer. They produced lateral diffusion coefficients D of 1.2 ± 0.05 × 10 -9 cm 2/s for C6-NBD-PC and 2.8 ± 0.3 × 10 -9 cm 2/s for C6-NBD-PE, when the two probes were located in the outer leaflet of the plasma membrane, just after cell labeling at 0°C. After cell incubation for one hour at 20°C, i.e., when C6-NBD-PC was still in the outer leaflet whereas C6-NBD-PE was translocated in the inner leaflet, D was observed to slightly increase for C6-NBD-PC ( D = 1.9 ± 0.06 × 10 -9 cm 2/s) and to greatly increase by at least a factor of 3 for C6-NBD-PE ( D = 9.1 ± 0.9 × 10 -9 cm 2/s). These results show that the plasma membrane of bovine aortic endothelial cells is equipped with a protein-dependent and energy-mediated phosphatidylethanolamine translocase activity and that the lateral diffusion rate of this phospholipid is much faster in the inner than in the outer leaflet of the lipid bilayer, thus indicating large differences in the fluidity of the two halves of this membrane.