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The capacity of ceramides to modify the permeability barrier of cell membranes has been explored. Membrane efflux induced either by in situgenerated 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 (C2-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 ≈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.

When vesicles composed of an equimolar mixture of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and cholesterol are treated with phospholipase C, phospholipid hydrolysis occurs without major changes in vesicle architecture. In the same way, addition of sphingomyelinase leads only to sphingomyelin cleavage. However, when both enzymes are added together, their joint hydrolytic activities give rise to leakage-free vesicle aggregation, lipid mixing, and aqueous contents mixing, i.e.vesicle fusion. The contribution of both enzymes is unequal, the main role of sphingomyelinase being the production of relatively large amounts of ceramide that will facilitate the lamellar-to-nonlamellar transition in the formation of the fusion pore, whereas phospholipase C provides mainly a localized, asymmetric, high concentration of diacylglycerol that constitutes the trigger for the fusion process. The lipidic end-products of both enzymes cooperate in destabilizing and fusing the membranes in a way that is never achieved through the action of any of the enzymes individually, nor by the products themselves when premixed with the other lipids during liposome preparation. Thus the enzymes appear to be coupled through their reaction products. This is the first observation of membrane fusion induced by the concerted activities of two enzymes. Besides, considering that both diacylglycerol and ceramide are important metabolites involved in cell signaling, it may also provide new ideas in the exploration of “cross-talk” phenomena between different signal transduction pathways.