Micropipette aspiration of human erythrocytes induces echinocytes via membrane phospholipid translocation

When a discocytic erythrocyte (RBC) was partially aspirated into a 1.5-mu m glass pipette with a high negative aspiration pressure (Delta P = -3.9 kPa), held in the pipette for 30 s (holding time, t(h)), and then released, it underwent a discocyte-echinocyte shape transformation. The degree of shape transformation increased with an increase in t(h). The echinocytes recovered spontaneously to discocytes in similar to 10 min, and there was no significant difference in recovery time at 20.9 degrees C, 29.5 degrees C, and 37.4 degrees C, respectively. At 11 degrees C the recovery time was significantly elevated to 40.1 +/- 6.7 min. At 20.9 degrees C the shape recovery time varied directly with the isotropic RBC tension induced by the pipetting. Sodium orthovanadate (vanadate, 200 mu M), which inhibits the phospholipid translocase, blocks the shape recovery. Chlorpromazine (CP, 25 mu M) reversed the pipette-induced echinocytic shape to discocytic in <2 min, and the RBC became a spherostomatocyte-II after another 30 min. It was hypothesized that the increase in cytosolic pressure during the pipette aspiration induced an isotropic tension in the RBC membrane followed by a net inside-to-outside membrane lipid translocation. After a sudden release of the aspiration pressure the cytosolic pressure and the membrane tension normalized immediately, but the translocated phospholipids remained temporarily ''trapped'' in the outer layer, causing an area excess and hence the echinocytic shape. The phospholipid translocase activity, when not inhibited by vanadate, caused a gradual return of the translocated phospholipids to the inner layer, and the RBC shape recovered with time.