Relationships between the surface exposure of acidic phospholipids and cell fusion in erythrocytes subjected to electrical breakdown

Abstract

The procoagulant activity of human erythrocytes, which provides a measure of the translocation of acidic phospholipids from the inner to the outer monolayer of the plasma membrane, has been compared with the percentage cell fusion in experiments on the effects of electrical breakdown pulses under differing experimental conditions. After treatment with breakdown pulses of 20 μs or longer (5 kV cm−1), the plasma membranes of erythrocytes in 250 mM sucrose exhibited an almost complete loss of asymmetry with respect to acidic phospholipids. As the breakdwon voltage was increased from 2 to 5 kV cm−1 (with breakdown pulses of 99 μs), the surface exposure of acidic phospholipids and cell fusion increased approximately in parallel. Furthermore, with 99 μs pulses and a voltage of 3 kV cm−1, a decrease in the osmolarity from 250 to 150 mM of the source medium was accompanied by an increase in both the surface exposure of acidic phospholipids and the extent of cell fusion. Breakdwon pulses of 2–5 μs were sufficient to cause a marked loss of asymmetry, but no cell fusion was observed unless the pulse length was at least 20 μs. Kinetic experiments indicated that exposure of the acidic phospholipids at the cell surface was more likely to be due to a direct effect of the electric field pulses on plasma membrane structure than to secondary effects, such as the action of endogenous proteinases on the membrane skeleton. It seems possible that a localised, surface exposure of acidic phospholipids may contribute to the ‘long-lived fusogenic state’ (Sowers, A.E. (1986) J. Cell Biol. 102, 1358–1362) and the ‘transient permeant structures’ (Teissié, J. and Rols, M.P. (1986) Biochem. Biophys. Res. Commun. 140, 258–266) than enable cell fusion to occur when contact between cells is established after they have been subjected to field pulses. Our observations alos provide circumstantial support for the concept that changes in the phospholipid asymmetry of membrane may be important in physiologically-occurring instances of biomembrane fusion.