DRUG-INDUCED TRANSMEMBRANE LIPID SCRAMBLING IN ERYTHROCYTES AND IN LIPOSOMES REQUIRES THE PRESENCE OF POLYANIONIC PHOSPHOLIPIDS

The asymmetric transmembrane distribution of phospholipids between the two bilayer halves of erythrocyte can be modified upon addition of ca tionic amphiphilic drugs, such as chlorpromazine or verapamil. We stud ied this phenomenon in erythrocytes and in lipid vesicles using spin-l abelled analogues of the endogenous phospholipids. The extent of the r apid disappearance of the analogues from the erythrocyte outer leaflet depended on the concentration of the drug. Up to 40% of spin-labelled sphingomyelin moved to the inner erythrocyte leaflet in 10 min in the presence of 1.5 mM chlorpromazine. Verapamil or vinblastine gave simi lar results. On the other hand, the inside-outside movement of the ami nophospholipid analogues was less evident, and did not exceed 10%. Thi s apparent discrepancy between inward and outward movements could resu lt from the formation of an endovesicle which is known to occur upon d rug addition at high concentration. A fraction of lipids would be trap ped in the intravesicular leaflet, corresponding to the cell outer lea flet, and be inaccessible both from the cytoplasm and the extracellula r medium. In cells submitted to a metabolic depletion of cellular ATP the intensity of the scrambling induced by the amphipaths was drastica lly lowered. We attribute this effect to the important reduction of th e membrane content in phosphatidylinositol-4,5-bisphosphate (PIP2). Th e involvement of the latter lipid in triggering scrambling was partly confirmed by experiments carried out with artificial membranes. Indeed , in large unilamellar vesicles PIP, is required in order to obtain a rapid redistribution of phospholipids between the two leaflets upon ad dition of drugs. However, the extent of phospholipid redistribution wa s limited to 15-20%. This redistribution was also induced when the ves icle membrane contained di-anionic phospholipids (phosphatidylinositol -4-monophosphate or diphosphatidylglycerol), but did not occur when it contained mono-anionic phospholipids (phosphatidylserine or phosphati dylinositol). Some drugs such as methochlorpromazine, active in artifi cial membranes, were ineffective in erythrocyte membranes, probably be cause they could not cross the membrane and reach PIP, molecules at th e cytoplasmic leaflet.