This study examines the relationship between cellular sphingomyelin content and the distribution of unesterified cholesterol between the plasma-membrane pool and the putative intracellular regulatory pool. The sphingomyelin content of cultured human skin fibroblasts was reduced by treatment of intact cells with extracellularly added neutral sphingomyelinase, and subsequent changes in the activities of cholesterol-metabolizing enzymes were determined. Exposure of fibroblasts to 0.1 unit of sphingomyelinase/ml for 60 min led to the depletion of more than 90% of the cellular sphingomyelin, as determined from total lipid extracts. In a time-course study, it was found that within 10 min of the addition of sphingomyelinase to cells, a dramatic increase in acyl-CoA:cholesterol acyltransferase activity could be observed, whether measured from the appearance of plasma membrane-derived [3H]cholesterol or exogenously added [14C]oleic acid, in cellular cholesteryl esters. In addition, the cholesteryl ester mass was significantly higher in sphingomyelin-depleted fibroblasts at 3 h after exposure to sphingomyelinase compared with that in untreated fibroblasts [7.1 +/- 0.4 nmol of cholesterol/mg equivalents of esterified cholesterol compared with 4.2 +/- 0.1 nmol of cholesterol/mg equivalents of cholesteryl ester in control cells (P less than 0.05)]. The sphingomyelin-depleted cells also showed a reduction in the rate of endogenous synthesis of cholesterol, as measured by incorporation of sodium [14C]acetate into [14C]cholesterol. These results are consistent with a rapid movement of cholesterol from sphingomyelin-depleted plasma membranes to the putative intracellular regulatory pool of cholesterol. This mass movement of cholesterol away from the plasma membranes presumably resulted from a decreased capacity of the plasma membranes to solubilize cholesterol, since sphingomyelin-depleted cells also had a decreased capacity to incorporate nanomolar amounts of [3H]cholesterol from the extracellular medium, as compared with control cells. These findings confirm previous assumptions that the membrane sphingomyelin content is an important determinant of the overall distribution of cholesterol within intact cells.

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This review is focused on the formation of lateral domains in model bilayer membranes, with an emphasis on sphingolipids and their interaction with cholesterol. Sphingolipids in general show a preference for partitioning into ordered domains. One of the roles of cholesterol is apparently to modulate the fluidity of the sphingolipid domains and also to help segregate the domains for functional purposes. Cholesterol shows a preference for sphingomyelin over phosphatidylcholine with corresponding acyl chains. The interaction of cholesterol with different sphingolipids is largely dependent on the molecular properties of the particular sphingolipid in question. Small head group size clearly has a destabilizing effect on sphingolipid/cholesterol interaction, as exemplified by studies with ceramide and ceramide phosphoethanolamine. Ceramides actually displace sterol from ordered domains formed with saturated phosphatidylcholine or sphingomyelin. The N-linked acyl chain is known to be an important stabilizer of the sphingolipid/cholesterol interaction. However, N-acyl phosphatidylethanolamines failed to interact favorably with cholesterol and to form cholesterol-enriched lateral domains in bilayer membranes. Glycosphingolipids also form ordered domains in membranes but do not show a strong preference for interacting with cholesterol. It is clear from the studies reviewed here that small changes in the structure of sphingolipids alter their partitioning between lateral domains substantially.

Received April 22, 1990; revision accepted October 30, 1990. when cells are loaded with cholesterol 13 -48 or when the rate of cell proliferation is inhibited. 9 These similar specificity and regulatory properties suggest that the 110-kd binding protein is a component of the cell-surface HDL binding sites on intact cells. Recent studies from our laboratory have provided evidence that the cellular HDL binding sites represent receptors that mediate transport of excess intracellular cholesterol from cells. Incubation of cholesterol-loaded cells with HDL3 stimulates translocation of radiolabeled sterol from intracellular pools to the plasma membrane and into the culture medium. 10 -11 This stimulation appears to require the interaction of HDL, with cell-surface binding sites, since modification of HDL3 with tetranitromethane (TNM) reduces its ability both to bind to cells 12 " 13 and to stimulate translocation and efflux of intracellular sterol. 1011 Because TNM treatment causes extensive covalent CTOss-linking of apos to other apos and phospholipids, 12 -13 it could not be determined from these studies whether modification of apos, lipids, or particle conformations accounted for the reduction in either cell-surface binding or sterol transport. Evidence that lipids rather than apos mediate cell-surface binding of HDL particles was provided by Tabas and Tall 14 in a study showing that trypsin treatment of HDL, failed to impair its ability to interact with cells.

To understand the structural basis for the apparent strong interaction between cholesterol and sphingomyelin (SPM), we have synthesized an analogue of SPM, 3-deoxy-2-O-stearoyl-SPM, in which an ester-linked acyl chain replaces the amide-linked acyl chain at C-2 and a hydrogen replaces the hydroxy group at C-3. We have compared the behavior of this analogue with that of 3-deoxy-N-stearoyl-SPM in monolayers and vesicles, both as pure phospholipids and in mixtures with cholesterol. The force-area isotherm of 3-deoxy-2-O-stearoyl-SPM was similar to that of 3-deoxy-N-stearoyl-SPM. The surface potential across the pure SPM monolayer at the air-water interface was larger for 3-deoxy-2-O-stearoyl-SPM than for 3-deoxy-N-stearoyl-SPM (about 430 mV and 330 mV, respectively, at 50 A2). The overall dipole moment of 3-deoxy-2-O-stearoyl-SPM was almost constant at 570 mD (between a mean molecular area range of 45-85 A2), whereas that of 3-deoxy-N-stearoyl-SPM was about 420 mD. Cholesterol appeared to be equally miscible in both SPM monolayers, as determined from the condensing effect cholesterol had on the lateral packing of the two SPMs. The oxidation of monolayer cholesterol by cholesterol oxidase was also determined using both SPMs. The stoichiometry at which free cholesterol clusters disappeared in monolayers, when going from high to low cholesterol content, was 2:1 (mol sterol/mol SPM) for both SPMs.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell membranes have a nonhomogenous lateral organization. Most information about such nonhomogenous mixing has been obtained from model membrane studies where defined lipid mixtures have been characterized. Various experimental approaches have been used to determine binary and ternary phase diagrams for systems under equilibrium conditions. Such phase diagrams are the most useful tools for understanding the lateral organization in cellular membranes. Here we have used the fluorescence properties of trans-parinaric acid (tPA) for phase diagram determination. The fluorescence intensity, anisotropy, and fluorescence lifetimes of tPA were measured in bilayers composed of one to three lipid components. All of these parameters could be used to determine the presence of liquid-ordered and gel phases in the samples. However, the clearest information about the phase state of the lipid bilayers was obtained from the fluorescence lifetimes of tPA. This is due to the fact that an intermediate-length lifetime was found in samples that contain a liquid-ordered phase and a long lifetime was found in samples that contained a gel phase, whereas tPA in the liquid-disordered phase has a markedly shorter fluorescence lifetime. On the basis of the measured fluorescence parameters, a phase diagram for the 1,2-dioleoyl-sn-glycero-3-phosphocholine/N-palmitoyl sphingomyelin/cholesterol system at 23 °C was prepared with a 5 mol % resolution. We conclude that tPA is a good fluorophore for probing the phase behavior of complex lipid mixtures, especially because multilamellar vesicles can be used. The determined phase diagram shows a clear resemblance to the microscopically determined phase diagram for the same system. However, there are also significant differences that likely are due to tPA's sensitivity to the presence of submicroscopic liquid-ordered and gel phase domains.

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.