J. Peter Slotte

Cholesterol interactions with phospholipids in membranes

Mammalian cell membranes are composed of a complex array of glycerophospholipids and sphingolipids that vary in head-group and acyl-chain composition. In a given cell type, membrane phospholipids may amount to more than a thousand molecular species. The complexity of phospholipid and sphingolipid structures is most likely a consequence of their diverse roles in membrane dynamics, protein regulation, signal transduction and secretion. This review is mainly focused on two of the major classes of membrane phospholipids in eukaryotic organisms, sphingomyelins and phosphatidylcholines. These phospholipid classes constitute more than 50% of membrane phospholipids. Cholesterol is most likely to associate with these lipids in the membranes of the cells. We discuss the synthesis and distribution in the cell of these lipids, how they are believed to interact with each other, and what cellular consequences such interactions may have. We also include a discussion about findings in the recent literature regarding cholesterol/phospholipid interactions in model membrane systems. Finally, we look at the recent trends in computer and molecular dynamics simulations regarding phospholipid and cholesterol/phospholipid behavior in bilayer membranes.

Hepatocellular uptake of 3H-dihydromicrocystin-LR, a cyclic peptide toxin

The cellular uptake of microcystin-LR, a cyclic heptapeptide hepatotoxin from the cyanobacterium Microcystis aeruginosa, was studied by means of a radiolabelled derivative of the toxin. 3H-dihydromicrocystin-LR. The uptake of 3H-dihydromicrocystin-LR was shown to be specific for freshly isolated rat hepatocytes whereas the uptake in the human hepatocarcinoma cell line Hep G2 as well as the mouse fibroblast cell line NIH-3T3, and the human neuroblastoma cell line SH-SY5Y, was negligible. By means of a surface barostat technique it was shown that the membrane penetrating capacity (surface activity) of microcystin-LR was low, indicating that the toxin requires an active uptake mechanism. The hepatocellular uptake of microcystin-LR could be inhibited in the presence of bile acid transport inhibitors such as antamanide (5 microM), sulfobromophthalein (50 microM) and rifampicin (30 microM). The uptake was also reduced in a concentration dependent manner when the hepatocytes were incubated in the presence the bile salts cholate and taurocholate. A complete inhibition of the hepatocellular uptake was achieved by 100 microM of either bile salt. The overall results indicate that the uptake of microcystin-LR is through the multispecific transport system for bile acids. This mechanism of cell entry would explain the previously observed cell specificity and organotropism of microcystin-LR.

Membrane properties of sphingomyelins

Sphingomyelin and phosphatidylcholine are important components in the external leaflet of cellular plasma membranes. In this review we compare the structure of these lipid molecules, with emphasis on the differences in hydrogen bonding capacity and membrane properties that arise from the small but significant differences in molecular structure. The membrane properties of sphingomyelins and the implications that these have, or might have, in biological membranes and for raft function are further discussed.

Sphingomyelin-cholesterol interactions in biological and model membranes.

Cholesterol and sphingomyelin are both important plasma membrane constituents in cells. It is now becoming evident that these two lipid classes affect each others metabolism in the cell to an extent that was not previously appreciated. It is the aim of this review to present recent data in the literature concerning both molecular and membrane properties of the two lipid classes, how they interact in membranes (both biological and model), and the consequences their mutual interaction have on different functional and metabolic processes in cells and lipoproteins.

INTERACTION OF CHOLESTEROL WITH SPHINGOMYELINS AND ACYL-CHAIN-MATCHED PHOSPHATIDYLCHOLINES : A COMPARATIVE STUDY OF THE EFFECT OF THE CHAIN LENGTH

In this study we have synthesized sphingomyelins (SM) and phosphatidylcholines (PC) with amide-linked or sn-2 linked acyl chains with lengths from 14 to 24 carbons. The purpose was to examine how the chain length and degree of unsaturation affected the interaction of cholesterol with these phospholipids in model membrane systems. Monolayers of saturated SMs and PCs with acyl chain lengths above 14 carbons were condensed and displayed a high collapse pressure ( approximately 70 mN/m). Monolayers of N-14:0-SM and 1(16:0)-2(14:0)-PC had a much lower collapse pressure (58-60 mN/m) and monounsaturated SMs collapsed at approximately 50 mN/m. The relative interaction of cholesterol with these phospholipids was determined at 22 degreesC by measuring the rate of cholesterol desorption from mixed monolayers (50 mol % cholesterol; 20 mN/m) to beta-cyclodextrin in the subphase (1.7 mM). The rate of cholesterol desorption was lower from saturated SM monolayers than from chain-matched PC monolayers. In SM monolayers, the rate of cholesterol desorption was very slow for all N-linked chains, whereas for PC monolayers we could observe higher desorption rates from monolayers of longer PCs. These results show that cholesterol interacts favorably with SMs (low rate of desorption), whereas its interaction (or miscibility) with long chain PCs is weaker. Introduction of a single cis-unsaturation in the N-linked acyl chain of SMs led to faster rates of cholesterol desorption as compared with saturated SMs. The exception was monolayers of N-22:1-SM and N-24:1-SM from which cholesterol desorbed almost as slowly as from the corresponding saturated SM monolayers. The results of this study suggest that cholesterol is most likely capable of interacting with all physiologically relevant (including long-chain) SMs present in the plasma membrane of cells.

Effects of sphingomyelin degradation on cell cholesterol oxidizability and steady-state distribution between the cell surface and the cell interior

This study addresses questions related to (i) the distribution of cholesterol between the cell surface and intracellular membranes in cultured fibroblasts and (ii) the effects of plasma membrane sphingomyelin on this distribution. Cholesterol oxidase (Streptomyces sp.) converts cell cholesterol to cholestenone and was used in this study to probe the cellular distribution of cholesterol. The availability of cell cholesterol for oxidation by cholesterol oxidase was markedly influenced by the presence of sphingomyelin. In native, glutaraldehyde-fixed fibroblasts only about 20% of the cell cholesterol was oxidized under our experimental conditions. However, degradation of cell sphingomyelin with sphingomyelinase (Staphylococcus aureus) markedly enhanced the oxidation of cell surface cholesterol in glutaraldehyde-fixed fibroblasts. About 90% of the total unesterified cholesterol could be oxidized to cholestenone in confluent, sphingomyelin-depleted fibroblasts. These results suggest that about 90% of the unesterified cholesterol was at the cell surface in these cells. It was also observed that degradation of cell sphingomyelin exerted a dramatic effect on the distribution of cell cholesterol between the cell surface and intracellular membranes. Within 90 min after hydrolysis of cell sphingomyelin, about 30% of the total cell-associated unesterified cholesterol was transported from a cholesterol oxidase-susceptible pool to an oxidase-resistant pool. Together with the redistribution of cell cholesterol after sphingomyelin degradation, a marked enhancement of the endogenous cholesterol esterification reaction was observed. We conclude that the degradation of plasma membrane sphingomyelin resulted in a new apparent steady-state distribution of cellular cholesterol, with less cholesterol in the plasma membrane and more in intracellular membranes. It therefore appears that sphingomyelin is a major determinant of the distribution of cholesterol in intact cells.

Biological functions of sphingomyelins

Sphingomyelin (SM) is a dominant sphingolipid in membranes of mammalian cells and this lipid class is specifically enriched in the plasma membrane, the endocytic recycling compartment, and the trans Golgi network. The distribution of SM and cholesterol among cellular compartments correlate. Sphingolipids have extensive hydrogen-bonding capabilities which together with their saturated nature facilitate the formation of sphingolipid and SM-enriched lateral domains in membranes. Cholesterol prefers to interact with SMs and this interaction has many important functional consequences. In this review, the synthesis, regulation, and intracellular distribution of SMs are discussed. The many direct roles played by membrane SM in various cellular functions and processes will also be discussed. These include involvement in the regulation of endocytosis and receptor-mediated ligand uptake, in ion channel and G-protein coupled receptor function, in protein sorting, and functioning as receptor molecules for various bacterial toxins, and for non-bacterial pore-forming toxins. SM is also an important constituent of the eye lens membrane, and is believed to participate in the regulation of various nuclear functions. SM is an independent risk factor in the development of cardiovascular disease, and new studies have shed light on possible mechanism behind its role in atherogenesis.

Does cholesterol discriminate between sphingomyelin and phosphatidylcholine in mixed monolayers containing both phospholipids

The objective of this work was to examine the interaction of cholesterol with both phosphatidylcholines, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and sphingomyelins, N-oleoyl-D-sphingomyelin (O-SPM) or N-palmitoyl-D-sphingomyelin (P-SPM), in monolayers at an air/water interface. We used cholesterol oxidase to probe for the relative strength of sterol-phospholipid interaction, and fluorescence microscopy to visualize lateral domain formation in the mixed monolayers. The ternary mixed monolayers, which contained cholesterol, POPC, and O-SPM had a twofold higher average oxidation rate than the corresponding system containing DPPC and P-SPM. This difference in oxidation rate between saturated and unsaturated systems was observed irrespective of the ratio between phosphatidylcholine and sphingomyelin in the monolayer. With either the saturated or the unsaturated systems, however, the rate of oxidation was influenced by the ratio of phosphatidylcholine to sphingomyelin. As the monolayer content of phosphatidylcholine increased and the sphingomyelin content decreased correspondingly (to maintain a constant cholesterol-to-phospholipid molar ratio), an increase in the average oxidation rate was seen in both saturated and mono-unsaturated monolayer systems. The relationship between the rate of cholesterol oxidation and the phosphatidylcholine/sphingomyelin ratio was not linear, suggesting a preferential interaction of cholesterol with sphingomyelin even when phosphatidylcholine was present in the monolayer. The formation and stability of cholesterol-rich lateral (liquid-condensed) domains in the monolayers, as determined by monolayer fluorescence microscopy, was found to be highly influenced by the phospholipid class, the degree of acyl chain saturation, and by the ratio of phosphatidylcholine to sphingomyelin in the monolayer. The differences in cholesterol oxidation rates and lateral domain formation, as a function of the ratio of two phospholipids in the monolayers, apparently derived from differences in the hydrophobic interactions between the lipids.

How the molecular features of glycosphingolipids affect domain formation in fluid membranes

Glycosphingolipids, sphingomyelin and cholesterol are often all found in the detergent resistant fraction of biological membranes and are therefore recognized as raft components, but they do not necessarily co-localize in the same lateral domains. From cell biological studies it is evident that different sphingolipid species can be found in different lateral regions within the same cellular membrane. Biophysical studies have shown that their tendency to co-localize with each other and with other membrane components is largely governed by structural features of all lipids present. Glycosphingolipids form gel-phase like domains in fluid lipid bilayers. Sphingomyelin readily associates with cholesterol, forming liquid-ordered phase domains, but glycosphingolipids do not readily form cholesterol-enriched domains by themselves. However, mixed sphingomyelin- and glycosphingolipid-rich domains appear to incorporate cholesterol. Recent studies indicate that the ceramide backbone structure as well as the number of sugar units and presence of charge in the glycosphingolipid head group will influence the partitioning of these lipids between lateral membrane domains. The properties of the domains will be largely influenced by the presence of glycosphingolipids, which have very high melting temperatures. The lateral partitioning of glycosphingolipid molecular species has only recently been studied more intensively, and a lot remains to be done in this field of research.

Membrane Properties of D-erythro-N-acyl Sphingomyelins and Their Corresponding Dihydro Species

We have prepared acyl chain-defined D-erythro-sphingomyelins and D-erythro-dihydrosphingomyelins and compared their properties in monolayer and bilayer membranes. Surface pressure/molecular area isotherms of D-erythro-N-16:0-sphingomyelin (16:0-SM) and D-erythro-N-16:0-dihydrosphingomyelin (16:0-DHSM) show very similar packing properties, except that the expanded-to-condensed phase transition (crystallization) occurs at a lower surface pressure for 16:0-DHSM. The measured surface potential was generally about 100 mV less for 16:0-DHSM monolayers compared to 16:0-SM monolayers. The condensed domains (crystals) that formed in 16:0-SM monolayers as a function of compression displayed star-shaped morphology when viewed under an epifluorescence microscope. 16:0-DHSM monolayers did not form similar crystals upon compression. 16:0-DHSM was degraded much faster by sphingomyelinase from Staphylococcus aureus than 16:0-SM (10-fold difference in enzyme activity needed for comparable hydrolytic rate). Cholesterol desorption from 16:0-DHSM to cyclodextrin was slightly slower (approximately 20%) than the rate measured from 16:0-SM monolayers (at 60 mol % cholesterol). The bilayer melting temperature of 16:0-DHSM was 47.7 degrees C (DeltaH 8.3 kcal/mol) whereas it was 41.2 degrees C for 16:0-SM (DeltaH 8.1 kcal/mol). Cholesterol/16:0-DHSM bilayers (15 mol % sterol) had more condensed domains than comparable 16:0-SM bilayers, as evidenced from the quenching resistance of DPH in DHSM membranes. We conclude that cholesterol interacts more favorably with 16:0-DHSM and that the membranes are more condensed than comparable 16:0-SM-containing membranes.