Chlorpromazine inhibits vesiculation, alters phosphoinositide turnover and changes deformability of ATP-depleted RBCs

Abstract

To delineate further the underlying mechanism by which amphiphilic drugs can modulate vesicle release from human RBCs, we studied the effect of chlorpromazine on erythrocyte vesiculation induced by ATP depletion. This was correlated with turnover of the phosphoinositides as well as RBC deformability during the process since phosphoinositide metabolism may be involved in shape regulation of RBCs. Echinocytic shape transformation and subsequent vesiculation of RBCs, which commonly occur during ATP depletion, were inhibited by chlorpromazine. Furthermore, with a newly developed two-dimensional thin-layer chromatography separation of RBC membrane phospholipids, we showed that chlorpromazine significantly decreased the dephosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) in both ATP-depleted RBCs as well as in cells with partly maintained ATP levels. Concomitantly, there was a smaller increase in the relative amount of phosphatidylinositol. In addition, chlorpromazine also inhibited the decreased in RBC deformability as well as the shift of osmotic fragility that occurs during ATP depletion of erythrocytes.

Chlorpromazine modulates the morphological macro- and microstructure of endothelial cells

“…Immediately before the start of the investigations, the cells were washed carefully in Dulbecco's PBS (9.6 g/l, Sigma) containing 0.1% glucose adjusted to pH 7. 4 (22). At concentrations below 200 µM, it does not significantly affect the osmolarity or the pH of a PBS (22).…”

“…RECENT EVIDENCE INDICATES that endothelial cell plasma membrane vesiculation in the form of caveolae serves as a membrane reservoir during stretch of the endothelial cells and possibly for transendothelial transport in certain domains of the cytoplasm (16,24,26). Previous studies have shown that the amphipathic, cationic, and antipsychotic drug chlorpromazine (CP) modulates the phospholipid bilayer balance by intercalation into the inner leaflet of the red blood cell plasma membrane (4). The drug may reach both sides of the membrane because it can cross the membrane, but, thereafter, CP selectively inserts into the inner half of the membrane bilayer by virtue of its cationic properties (1,4).…”

“…Previous studies have shown that the amphipathic, cationic, and antipsychotic drug chlorpromazine (CP) modulates the phospholipid bilayer balance by intercalation into the inner leaflet of the red blood cell plasma membrane (4). The drug may reach both sides of the membrane because it can cross the membrane, but, thereafter, CP selectively inserts into the inner half of the membrane bilayer by virtue of its cationic properties (1,4). According to the bilayer couple hypothesis (28), one-sided intercalation leads to expansion of the inner membrane layer relative to the outer layer causing the red blood cells to adopt a cupped shape.…”

“…According to the bilayer couple hypothesis (28), one-sided intercalation leads to expansion of the inner membrane layer relative to the outer layer causing the red blood cells to adopt a cupped shape. Because CP accumulates at the inner leaflet of the membrane bilayer, it may also modulate the activity of membrane-associated enzymes, located in the inner half of the bilayer, that are involved in phosphoinositide turnover (4,27). By changing the membrane environment and the surface charge density (1,14), the inward shift of phospholipids occurs faster than the diffusion outwards (27).…”

“…By changing the membrane environment and the surface charge density (1,14), the inward shift of phospholipids occurs faster than the diffusion outwards (27). Thereby, CP induces a new bilayer asymmetry and an inner-layer expansion that causes a bilayer buckling without changing the membrane protein composition or affecting ATP levels (4). Thus CP causes a time-and concentrationdependent concave plasma membrane bending that leads to macroscopic morphological changes in red blood cells toward stomatocytic cell shapes (2,12,18,21).…”