Divalent cations, phospholipid asymmetry and osmotic swelling in electrically-induced lysis, cell fusion and giant cell formation with human erythrocytes

Abstract

We have previously reported that acidic phospholipids are exposed at the surface of human erythrocytes when the cells are subjected to electrical breakdown. It has now been shown that the prothrombinase assay, which was used previously for the determination of acidic phospholipids, is specific for phosphatidylserine under the conditions of our experiments. In the light of this finding, we have investigated and characterised factors that govern cell lysis, cell fusion, and the formation of giant cells induced by electrical breakdown with human erythrocytes in media of low ionic strength. Divalent cations (1.1 mM) protected the cells against haemolysis, in the order Mn 2+{rmCa 2+ > Ba 2+ > Mg 2+ > Zn 2+, whereas about 99% of the cells cells lysed immediately on breakdown in the presence of Na + or K + (2.1 mM), or A1 3+ (0.95 mM). The lengths of pearl chains of fused erythrocytes formed was similarly greatest with Mn 2+ and decreased progressively with Ba 2+, Zn 2-, Ca 2+ and Mg 2+. No cell fusion occurred with Na +, K +, or A1 3+. It is suggested that interactions with phosphatidylserine, which is exposed at the cell surface by electrical breakdown, may enable Mn 2-, Ba 2+ and Ca 2+ ions to inhibit cell lysis (via membrane resealing) and facilitate cell fusion. Following electrically-induced cell fusion, erythrocytes round-up into giant cells. It has previously been proposed that Ca 2+ ions accelerate the rounding-up process. However, data are presented which show that, as with erythrocytes treated with Sendal virus, the formation of rounded, giant cells following cell fusion depends on the osmotic swelling properties of permeabilised erythrocytes. Osmotic swelling may also have induced any hemi-fused cells present to fuse completely. Zn 2+ ions anomalously enabled erythrocytes to round-up very rapidly into giant cells following electrical breakdown. This phenomenon may result from an interaction of Zn 2+ ions with cysteine groups in membrane proteins, which decreases the immediate loss of ions that occurs when erythrocytes are subjected to electrical breakdown in low-ionic-strength media.