Abstract
The capacity of ceramides to modify the permeability barrier of cell membranes has been explored. Membrane efflux induced either by in situ generated ceramides (through enzymatic cleavage of sphingomyelin) or by addition of ceramides to preformed membranes has been studied. Large unilamellar vesicles composed of different phospholipids and cholesterol, and containing entrapped fluorescent molecules, have been used as a system to assay ceramide-dependent efflux. Small proportions of ceramide (10 mol % of total lipid) that may exist under physiological conditions of ceramide-dependent signaling have been used in most experiments. When long chain (egg-derived) ceramides are used, both externally added or enzymatically produced ceramides induce release of vesicle contents. However, the same proportion of ceramides generated by sphingomyelinase induce faster and more extensive efflux than when added in organic solution to the preformed vesicles. Under our conditions 10 mol % of N-acetylsphingosine (C(2)-ceramide) did not induce any efflux. On the other hand, sphingomyelinase treatment of bilayers containing 50 mol % sphingomyelin gave rise to release of fluorescein-derivatised dextrans of molecular mass approximately 20 kDa, i.e. larger than cytochrome c. These results have been discussed in the light of our own previous data (Ruiz-Argüello, M. B., Basañez, G., Goñi, F. M., and Alonso, A. (1996) J. Biol. Chem. 271, 26616-26621) and of the observations by Siskind and Colombini (Siskind, L. J., and Colombini, M. (2000) J. Biol. Chem. 275, 38640-38644). Our spectroscopic observations appear to be in good agreement with the electrophysiological studies of the latter authors. Furthermore, some experiments in this paper have been designed to explore the mechanism of ceramide-induced efflux. Two properties of ceramide, namely its capacity to induce negative monolayer curvature and its tendency to segregate into ceramide-rich domains, appear to be important in the membrane restructuring process.
Highlights
Ceramides have emerged in the last decade as important messengers in cell signaling involved among others, in pro
Ceramide-induced release of aqueous contents from liposomes and resealed erythrocyte ghosts was first observed by Ruiz-Arguello et al [3], who induced in situ generation of ceramide by treating the sphingomyelincontaining model or cell membranes with bacterial sphingomyelinase
Our results show that the presence of ceramides can allow the efflux of large molecules through membranes, that both in situ generation and external addition of ceramides can induce release, and that electrophysiological detection of pore formation correlates with release of vesicular contents
Summary
Ceramides have emerged in the last decade as important messengers in cell signaling involved among others, in pro-. We have attempted to answer a number of questions that remain open after publication of the above data, in particular the possibility of inducing the release of large molecules (the size of cytochrome c) by ceramide, the existence of common aspects in the efflux induced by in situ enzyme-generated and by externally added ceramide, and the correlation between electrophysiological obervations of channel formation and biochemical/biophysical detection of release of vesicle or cell contents. Our results show that the presence of ceramides can allow the efflux of large molecules (molecular mass Ϸ20 kDa) through membranes, that both in situ generation and external addition of ceramides can induce release, and that electrophysiological detection of pore formation correlates with release of vesicular contents ( with significant differences in the time scale of detection of the phenomena). The role of the molecular geometry of ceramides, and of their immiscibility with other lipids in the mechanism of membrane permeabilization are supported by the experimental data
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