Ceramide and cholesterol interactions in phospholipid membranes: A 2H NMR study

Sphingolipids constitute a significant fraction of cellular plasma membrane lipid content. Among sphingolipids, ceramide levels are usually very low. However, in some cell processes like apoptosis, cell membrane ceramide levels increase markedly due to activation of enzymes like sphingomyelinase. This increase can change the physical state of the membrane by promoting molecular order and inducing solid ordered (So) phase domains. This effect has been observed in a previous 2H NMR study on membranes consisting of palmitoyl sphingomyelin (PSM) and palmitoyl ceramide (PCer). Cholesterol (Chol), is also present at high concentrations in mammalian plasma membranes and has a favorable interaction with sphingomyelin (SM), together forming domains in the liquid ordered (Lo) phase in model membranes. There are reports that Chol is able to displace ceramide (Cer) in SM bilayers and abolish the So phase domains formed by SM:Cer. This ability of Chol appears to be concentration dependent; in membranes with low Chol and high Cer contents, So phase domains hypothesized to be rich in Cer coexist with the continuous fluid phase of the membrane. Here, first we study the effect of increasing PCer concentration in PSM:Chol bilayers, using 2H NMR. Chol:PCer mol ratios were 3:1, 3:2 and 3:3, at a fixed 7:3 PSM:cholesterol mol ratio. Both PSM and PCer were monitored, in separate samples, for changes in their physical state by introducing a perdeuterated palmitoyl chain in either molecule. Second, we investigate the effect of replacing PSM with DPPC to test the influence on membrane phase behavior of replacing sphingosine with a palmitoylated glycerol backbone. Third, we explore the effect of adding an unsaturated lipid present at a high level in plasma membranes, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), to the PSM:Chol:PCer 7:3:3 lipid mixtures. This was done to study an approximate cell membrane outer leaflet mimetic lipid mixture. We found that PCer induces highly stable So phase domains in PSM:Chol, DPPC:Chol and POPC:PSM:Chol bilayers. This effect is most pronounced in bilayers with Chol:PCer 1:1 molar ratios, and below 40 oC. PCer is more effective in ordering PSM:Chol bilayers than analogous bilayers composed of DPPC:Chol.