Shear stress induces a time- and position-dependent increase in endothelial cell membrane fluidity.

Blood flow-associated shear stress may modulate cellular processes through its action on the plasma membrane. We quantified the spatial and temporal aspects of the effects of shear stress (tau) on the lipid fluidity of 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC(16)(13)]-stained plasma membranes of bovine aortic endothelial cells in a flow chamber. A confocal microscope was used to determine the DiI diffusion coefficient (D) by fluorescence recovery after photobleaching on cells under static conditions, after a step-tau of 10 or 20 dyn/cm(2), and after the cessation of tau. The method allowed the measurements of D on the upstream and downstream sides of the cell taken midway between the respective cell borders and the nucleus. In <10 s after a step-tau of 10 dyn/cm(2), D showed an upstream increase and a downstream decrease, and both changes disappeared rapidly. There was a secondary, larger increase in upstream D, which reached a peak at 7 min and decreased thereafter, despite the maintenance of tau. D returned to near control values within 5 s after cessation of tau. Downstream D showed little secondary changes throughout the 10-min shearing, as well as after its cessation. Further investigations into the early phase, with simultaneous measurements of upstream and downstream D, confirmed that a step-tau of 10 dyn/cm(2) elicited a rapid (5-s) but transient increase in upstream D and a concurrent decrease in downstream D, yielding a significant difference between the two sites. A step-tau of 20 dyn/cm(2) caused D to increase at both sites at 5 s, but by 30 s and 1 min the upstream D became significantly higher than the downstream D. These results demonstrate shear-induced changes in membrane fluidity that are time dependent and spatially heterogeneous. These changes in membrane fluidity may have important implications in shear-induced membrane protein modulation.

Micropipette aspiration of human erythrocytes induces echinocytes via membrane phospholipid translocation.

When a discocytic erythrocyte (RBC) was partially aspirated into a 1.5-microns glass pipette with a high negative aspiration pressure (delta P = -3.9 kPa), held in the pipette for 30 s (holding time, th), and then released, it underwent a discocyte-echinocyte shape transformation. The degree of shape transformation increased with an increase in th. The echinocytes recovered spontaneously to discocytes in approximately 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 microM), which inhibits the phospholipid translocase, blocks the shape recovery. Chlorpromazine (CP, 25 microM) 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.

Folding of red blood cells in capillaries and narrow pores.

The geometric features of red blood cells in narrow channels in vivo and in vitro were studied by electron microscopy. In rabbit myocardial capillaries about half of the red cells were folded. In polycarbonate filters with pore diameters of 2.2-4.5 microns approximately one third of the trapped red blood cells were folded. The frequency of folding did not depend on the applied pressure, which ranged from 0.1 to 8.0 cm H2O. The folding of the red blood cells in filter pores was used to estimate the bending stiffness of the membrane. An analysis based on the large deformation theory of bending of an elastic sheet was developed. Using pressures of 0.2 and 1.0 cm H2O, the bending stiffness of human red cell membranes was estimated to be approximately 2.4 - 11.6 x 10(-12) dyn-cm, which is in good agreement with other methods. A limiting radius of curvature of about 85 nm was found at higher pressures.