Inhibition and stimulation of phospholipid scrambling activity. Consequences for lipid asymmetry, echinocytosis, and microvesiculation of erythrocytes

An increase of the intracellular Ca2+ concentration in erythrocytes is know n to activate rapid nonspecific bidirectional translocation of membrane-ins erted phospholipid probes and to decrease the asymmetric distribution of en dogenous membrane phospholipids. These scrambling effects are now shown to be suppressed by pretreatment of cells with the essentially impermeable rea gents 4,4 ' -diisothiocyanostilbene-2,2 ' -disulfonic acid and 2,4,6-trinit robenzenesulfonate. The inhibitory effects are no loner observed during ren ewed activation of scrambling following a first transient activation by Ca2 +. Assuming the involvement of the human scramblase, this suggests a confor mational alteration of this protein during activation by Ca2+. Marked suppr ession of scrambling activity is also observed in cells pretreated with the disulfide reducing agent dithioerythritol which can be reverted by the SH oxidizing agent diamide. This indicates the importance of intramolecular an d/or intersubunit disulfide bonds for the function of the scramblase. On th e other hand, treatment of cells with the SH reagents N-ethylmaleimide and phenylarsine oxide enhances Ca2+-activated scrambling and diminution of asy mmetry of membrane phospholipids: This suggests an allosteric connection of several protein SH groups to the translocation mechanism. The inhibitors r etain their strong suppressive effects. Besides covalent modification, addi tion of oligomycin highly stimulates and addition of clotrimazole suppresse s the Ca2+-activated translocation. No evidence for a role of the ATP-bindi ng cassette transporter ABCA1 in the Ca2+-activated outward translocation i s obtained. Suppression of phospholipid scrambling by dithioerythritol inhi bits Ca2+-induced spheroechinocytosis and reduces the extent of subsequent microvesiculation. Scrambling of endogenous phospholipids is proposed to in duce echinocytosis and to have only a stimulatory effect on microvesiculati on.