Involvement of lipid rafts of rabbit red blood cells in morphological changes induced by methylated...

Lipid rafts on cell membranes have heterogeneity such as cholesterol-rich microdomains and sphingolipids-rich microdomains. We previously reported that beta-cyclodextrin (beta-CyD) induced morphological changes of red blood cells (RBC) from discocyte to stomatocyte, possibly due to extraction of cholesterol from cholesterol-rich lipid rafts of RBC membranes. In this study, the effects of methyl-beta-cyclodextrin (M-beta-CyD) and 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CyD) on lipid rafts and morphological changes in rabbit RBC (RRBC) were examined, compared to those of beta-CyD. In sharp contrast to beta-CyD, M-beta-CyD and DM-beta-CyD induced morphological changes of RRBC from discocyte to echinocyte. At pre-hemolytic concentrations of beta-CyDs, M-beta-CyD and DM-beta-CyD strongly released cholesterol from cholesterol-rich lipid rafts, compared to beta-CyD. Meanwhile, the lowering effects of DM-beta-CyD on fluorescent sphingomyelin analogue in sphingolipids-rich lipid rafts were more potent than those of beta-CyD and M-beta-CyD. The magnitude of the abilities of M-beta-CyD and DM-beta-CyD to extract membrane constituents was higher than that of beta-CyD, consistent with that of hemolytic activity. Furthermore, DM-beta-CyD and M-beta-CyD, not beta-CyD, lowered the amount of proteins in cholesterol-rich lipid rafts of RRBC. These results suggest that higher hemolytic activity and morphological changes from discocyte to echinocyte in RRBC induced by M-beta-CyD and DM-beta-CyD may be due to the extraction of both cholesterol and proteins from cholesterol-rich lipid rafts of RRBC, although DM-beta-CyD may interact with sphingolipids-rich lipid rafts on RRBC membranes only slightly.