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Chapter 10 -Sphingomyelin Interactions in Cells-Effects on

J. Peter Slotte

1. INTRODUCTION

Both cholesterol and sphingomyelin are important constituents of cellular plasma membranes. The molecules are chemically and functionally very different, yet they appear to be attracted to each other in the membrane compartment. It is the aim of this review to discuss how alterations in their membrane interactions may affect lipid homeostasis in cells, and to suggest a molecular explanation for their mutual affinity in membranes. Recent reviews dealing with the subcellular distribution and transport of cholesterol (Liscum and Dahl, 1992; Liscum and Faust, 1994; Liscum and Under• wood, 1995), with cellular lipid traffic (van Meer, 1989; Pagano, 1990; Voelker, 1991; Allan and Kallen, 1993), with transport and metabolism of sphingomyelin (Koval and Pagano, 1991), and with the role of in cell signaling (Kolesnick, 1991, 1994; Hannun and Bell, 1993; Hannun, 1994), may also be of interest to the reader.

2. CELLULAR DISTRIBUTION OF CHOLESTEROL

2.1. Cell Cholesterol Homeostasis

Cholesterol is an essential component of cellular plasma membranes in higher organisms. Cholesterol interacts with membrane and

J. Peter SloUe Department of Biochemistry and Pharmacy, Abo Akademi University, FIN 20520 Turku, Finland. Subcellular Biochemistry, Volume 28: Cholesterol: Its Functions and Metabolism in Biology and Medi• cine, edited by Robert Bittman. Plenum Press, New York. 1997.

R. Bittman (ed.), Cholesterol 277 © Plenum Press, New York 1997 278 J. Peter Slotte influences their physico-chemical properties. The important membrane proper• ties that are directly or indirectly influenced by membrane levels of cholesterol include solute permeability (for a review, see Yeagle, 1985), acyl chain mobility (GaIly et ai., 1976; Stockton and Smith, 1976, Yeagle, 1985) and lateral packing (Chapman et ai., 1969; Lund-Katz et ai., 1988; Smaby et ai., 1994). Cholesterol also has a marked influence on lateral phase separations (Smutzer and Yeagle, 1985), and on the effective free volume of membranes (Straume and Litman, 1987), two parameters that are directly related to the flexi• bility of membrane proteins (e.g., ion channels, ) and hence to their func• tion in membranes. Since cholesterol is such an essential and ubiquitous molecule in cells, most eucaryotic cells have retained the capacity to synthesize cholesterol from two• carbon precursor molecules. An important and early rate-limiting step in the biosynthesis of cholesterol is the conversion of ~-hydroxy-~-methylglutaryl-CoA to mevalonate, catalyzed by ~-hydroxy-~-methylglutaryl-CoA reductase (HMG• CoA reductase; Brown et ai., 1973). The regulation of HMG-CoA reductase in• cludes (long-term) transcriptional regulation, probably by oxygenated sterols, as well as inactivation/activation of the by an AMP-dependent protein kinase/phosphatase cascade (Beg et aI., 1978; Ingebritsen et ai., 1981; Brown and Goldstein, 1986; Mehrabian et aI., 1986; Gibson and Parker, 1987). An alternative source of cellular cholesterol is provided by low density lipoproteins (LDL), which bind to plasma membrane apo BIE receptors (Goldstein and Brown, 1977). The receptorlLDL complex is internalized by a process termed receptor-mediated endocytosis (Brown and Goldstein, 1986), in which the recep• tor ligand is transported to the lysosomal compartment for partial or complete degradation (Brown et aI., 1975). Unesterified cholesterol in the (released from the LDL particles or derived from the hydrolysis of LDL-derived cholesteryl esters) is transported directly to the plasma membrane compartment. Eventually, any excess of plasma membrane unesterified cholesterol may be trans• ported to the for conversion to cholesteryl esters (Tabas et aI., 1988). Rates of LDL internalization and cellular cholesterol esterification (by acyl-CoA:cholesterol acyl transferase; ACAT) are sensitive to cellular levels of unesterified cholesterol (Goldstein and Brown, 1977). An unregulated pathway for cellular uptake of lipoprotein-derived unesterified cholesterol involves the di• rect (surface) transfer of lipoprotein surface cholesterol to the cellular plasma membrane compartment. This pathway may contribute significantly to cell choles• terol accumulation when the extracellular level ofLDL is high (Slotte et ai., 1988). Since extrahepatic and nonsteroidogenic cells cannot metabolize cholesterol, save for the conversion of cholesterol to cholesteryl esters, routes must exist whereby cells can rid themselves of excess cholesterol. High-density lipoproteins have been shown to act as cholesterol acceptors, and initiate the reverse-cholesterol transport system first proposed by Glomset in 1968 (Glomset, 1968; Rothblat and Phillips, 1991). It is at present unclear which of the HDL subtypes are the best cho- Cholesterol-Sphingomyelin Interactions 279

Ie sterol acceptor particles, although their capacities to initiate cholesterol efflux from cells clearly differ (Rothblat et aI., 1992; Mahlberg and Rothblat, 1992; Davidson et at., 1994). Whereas the actual transfer of cholesterol from cellular plasma membranes to extracellular acceptor particles apparently do not require binding proteins (Johnson et aI., 1991), the interaction of HDL-like particles with specific cell surface binding proteins may lead to a regulated flow (translocation) of intracellular cholesterol to the cell surface (Slotte et aI., 1987; Mendez et aI., 1991; Porn et at., 1991a).

2.2. Subcellular Distribution of Cholesterol

Most of the cellular unesterified cholesterol can be found in the plasma mem• brane compartment. Depending on the cell type examined and the assay method used, plasma membranes have been reported to contain from about 50% to as much as 90% of the total cellular unesterified cholesterol (Lange, 1991; Warnock et aI., 1993). It appears that cells with an extensive recycling of plasma membranes through the en• docytic system have a larger fraction of intracellular cholesterol (Allan and Kallen, 1993). It is not surprising that cellular plasma membranes contain such a high con• centration of cholesterol, since several of the cellular cholesterol transport pathways are directed toward the cell surface. Newly synthesized cholesterol is transported vec• tori ally from the intracellular site of synthesis to the cell surface, where it appears with a transfer half-time of 10-18 min (DeGrella and Simoni, 1982; Lange et aI., 1991; Urbani and Simoni, 1990; Kaplan and Simoni, 1985). Exogenously derived choles• terol, entering the cell with LDL particles, is also quantitatively transported from lyso• somes to the cell surface, where it accumulates (Tabas et aI., 1988). Only when the solubilizing capacity of the plasma membrane compartment is saturated does the ex• cess cholesterol move to the endoplasmic reticulum for esterification by ACAT (Xu and Tabas, 1991). It is now established that the capacity of plasma membranes to solu• bilize cholesterol is largely a function of its sphingomyelin content (see section 5).

3. SYNTHESIS AND DISTRIBUTION OF SPHINGOMYELIN

3.1. Site of Sphingomyelin Synthesis in Cells

Sphingomyelin appears to be synthesized by the enzymatic transfer of the moiety of to (catalyzed by sphingo• synthase), thus yielding sphingomyelin and diacylglycerol (Voelker and Kennedy, 1982; Spence et at., 1983; Marggraf and Kanfer, 1984; Kishimoto, 1993). The direct transfer of CDP- to ceramide is apparently a pathway of minor significance in most cells (Voelker and Kennedy, 1982). There is currently a dispute regarding the major site of sphingomyelin synthesis, since synthetic activ• ities have been demonstrated (directly or indirectly) in several different cellular 280 J. Peter Slotte compartments (Futerman et aI., 1990; van Echten et aI., 1990; leckel et aI., 1990; Kallen et aI., 1993, 1994). In studies using subcellular fractionation techniques (rat ), it was observed that cis Golgi fractions accounted for the largest activity of sphingomyelin synthesis, whereas only a small fraction of the synthetic activity could be attributed to the plasma membrane fraction (Futerman et aI., 1990; leckel et aI., 1990). In baby hamster kidney cells, however, it was reported that a sub• stantial synthesis of sphingomyelin occurred in a compartment distinct from the cis/medial Golgi. This compartment was presumed to include the endocytic recy• cling pathway (Kallen et al., 1993, 1994). There is always the possibility that the predominant site of synthesis may differ from one cell type to another, as a differ• ent synthetic topology may be needed for functionally specialized cells. This is in• deed discussed in a recent report, where it was shown that the plasma and myelin membrane compartment accounted for at least half of the sphingomyelin synthase activity in oligodendrocytes (Vos et aI., 1995).

3.2. Subcellular Distribution of Sphingomyelin

The distribution of sphingomyelin mass among intracellular organelles has been examined using cell fractionation studies and sphingomyelinase degradation experiments. Results with both techniques suggest that more than half of the cel• lular sphingomyelin mass is confined to the plasma membranes (with most of it being in the exoleaflet; for a review, see Koval and Pagano, 1991). With fibro• blasts it was reported that as much as 90% of the total cell sphingomyelin mass was in the plasma membrane compartment (Lange, 1991). Again, as was the case with cholesterol distribution, it appears that cells with an extensive recycling of plasma membranes through the endocytic system also have a larger fraction of sphingomyelin in intracellular compartments (inaccessible to exogenously applied sphingomyelinase) (Allan and Kallen, 1993). The predominant exoleaflet orienta• tion of plasma membrane sphingomyelin apparently results from the topological orientation of sphingomyelin synthase, since sphingomyelin flip-flop from one leaflet to the other is very limited, and since a membrane translocase acting on sphingomyelin has not yet been observed.

4. EFFECTS OF SPHINGOMYELIN ON CELL CHOLESTEROL METABOLISM

4.1. Increased Sphingomyelin Levels versus Cholesterol Homeostasis

The older observations that there appeared to exist a positive correlation be• tween the contents of cholesterol and sphingomyelin mass in the membranes of rat liver hepatocytes (Patton, 1970), and the more recent data indicating colocal• ization of these lipid classes in the plasma membrane compartment (as well as in Cholesterol-Sphingomyelin Interactions 281 the exocytic cycling pathway), may suggest that one of these lipid classes affect the distribution of the other (directly or indirectly). Early evidence indicating that plasma membrane levels of sphingomyelin might affect the content of cholesterol in the membrane was obtained from sphingomyelin loading experiments in cells. The incorporation of sphingomyelin mass from liposomes into fibroblast mem• branes was observed to result in a reduced esterification of cell cholesterol, and to an increased formation of newly synthesized cholesterol (Gatt and Bierman, 1980; Kudchodkar et ai., 1983). Since both the ACAT-catalyzed esterification re• action and the biosynthesis of cholesterol are acutely regulated by the cellular level of unesterified cholesterol, the sphingomyelin loading experiments sug• gested that sphingomyelin incorporation directly led to a net flow of cholesterol from intracellular sites to the plasma membrane compartment. Plausibly, the in• corporation of sphingomyelin mass in the cellular membranes increased the ca• pacity of cells to solubilize cholesterol, which in tum resulted in the observed metabolic responses.

4.2. Decreased Sphingomyelin Levels versus Cholesterol Homeostasis

4.2.1. Effects on Cholesterol Esterification

Since an increased level of sphingomyelin mass in cell membranes led to a marked flow of cell cholesterol toward the cell surface, it seemed likely that a re• distribution in the opposite direction would occur in cells deprived of cell surface sphingomyelin. Experiments along these lines were first performed by Slotte and Bierman (1988), who showed that the degradation of sphingomyelin mass (with the aid of exogenous sphingomyelinase) in cultured fibroblasts led to a dramatic activation of the endogenous esterification of cholesterol. Since the ACAT• reaction in the endoplasmic reticulum is sensitive to the flow of substrate choles• terol, its activation strongly suggested that sphingomyelin degradation resulted in a flow of cholesterol from the cell surface into the substrate pool of ACAT. This pioneering study was subsequently followed by related studies with differ• ent cell types, in which similar effects of sphingomyelin degradation on the acti• vation of ACAT consistently have been observed (Gupta and Rudney, 1991; Stein et ai., 1992). The sphingomyelinase-induced activation of ACAT was not seen when cells were exposed to degradation products of sphingomyelin such as phos• phoryIcholine, ceramide, or (Slotte and Bierman, 1988; Gupta and Rudney, 1991).

4.2.2. Effects on Cholesterol Biosynthesis

In addition to affecting the rate of endogenous cholesterol esterification, the sphingomyelinase-induced cholesterol translocation also affected the rate of cho• lesterol biosynthesis. Slotte and Bierman (1988) originally demonstrated that the 282 J. Peter Slotte incorporation of sodium [14C]acetate into de novo sterols was reduced markedly in cells treated with sphingomyelinase as compared to control fibroblasts. Later, Gupta and Rudney (1991) extended these experiments to include rat intestinal epi• thelial cells, human skin fibroblasts, and HepG2 cells, and demonstrated that sphingomyelin degradation, in conjunction with the resulting cholesterol flow, di• rectly downr~gulated the activity of HMG-CoA reductase, the key regulatory en• zyme in cholesterol biosynthesis.

4.2.3. Effects on Steroidogenesis

In steroidogenic cells, cholesterol is converted to various steroid hormones by the action of cytochrome P450scc in the mitochondrial inner membrane. For this con• version to take place, cholesterol must first be transported from cellular storage sites (cholesteryl ester droplets, from which free cholesterol is released by hydrolysis; plasma membrane cholesterol pools) to the mitochondria (Bisgaier et ai., 1985; Free• man, 1989). Transfer of cholesterol from the outer mitchondrial membrane to the inner appears to involve a steroidogenic acute regulatory protein (Lin et ai., 1995). In our experiments with Leydig tumor cells, the degradation of cell surface sphingomyelin was observed to increase the formation of steroid hormones (Porn et ai., 1991b). These results were interpreted to demonstrate that a significant frac• tion of cell surface cholesterol, which was induced to translocate by the action of sphingomyelinase, was transferred to the mitochondrial compartment to serve as a substrate for cytochrome P450scc, to yield newly synthesized steroid hormones.

4.2.4. Cellular Recovery from Sphingomyelin Degradation

The recovery of cells from the effects of sphingomyelin degradation were tested with several different cell types (Porn and Slotte, 1990; Slotte et al., 1990). When cells that initially were exposed to sphingomyelinase (to yield sphingo• myelin degradation) were allowed to recover in the absence of exogenous sphingo• myelinase, a restoration of sphingomyelin mass was observed to take place. The rate of sphingomyelin resynthesis was, however, very different in different cell types. A very slow restoration of sphingomyelin levels was observed in human fibroblasts and SH-SY5Y neuroblastoma cells, whereas a much faster restoration was seen with BHK-21 cells. However, with all cell types tested, the restoration of sphingomyelin mass also restored cholesterol enrichment at the cell surface, and decreased the ac• tivation of the ACAT-reaction (Porn and Slotte, 1990; Slotte et ai., 1990).

4.2.5. Determination of Cellular Cholesterol Distribution in Sphingomyelin-Depleted Cells

In an attempt to quantitate cell surface cholesterol in whole cells, using a modified cholesterol oxidase assay (Slotte et ai., 1989), it was observed that the Cholesterol-Sphingomyelin Interactions 283 complete degradation of sphingomyelin mass from the external leaflet of the cel• lular plasma membrane invariably led to a rapid and substantial decrease of the fraction of cell cholesterol susceptible to oxidation by cholesterol oxidase (Slotte et al., 1989; Porn and Slotte, 1990; Slotte et al., 1990). This decrease in cell cho• lesterol oxidizability has been interpreted to indicate a substantial translocation of cell surface cholesterol to intracellular membranes. The extent of cholesterol translocation appear to differ from one cell type to another (between 20 and 60% of the cellular free cholesterol), and also within different lines of a similar cell type (i.e., human fibroblasts; J. P. Slotte, M. I. Porn, and A. S. Harmala, unpublished observations). A recent study, in which the cellular distribution of cholesterol was de• termined using filipin staining and fluorescence microscopy, showed that the filipin-staining pattern was not significantly different in control and sphingo• myelin-depleted cells (Porn and Slotte, 1995). These results do not invalidate previous findings about the sphingomyelinase-induced cholesterol translocation in cells (Slotte et aI., 1989; Porn and Slotte, 1990; Slotte et al., 1990), but do suggest, however, that the extent of cholesterol translocation is not as dramatic as suggested by the decreased cholesterol oxidation susceptibility. In light of both the filipin-staining results and the cholesterol oxidation results, it appears that sphingomyelin depletion results in a massive redistribution of cholesterol within the plasma membrane structure, but that only a smaller fraction is actu• ally transferred to intracellular sites (e.g., mitochondria and the endoplasmic reticulum).

4.2.6. Phosphatidylcholine Degradation Does Not Affect Cholesterol Homeostasis

To examine whether a degradation of other plasma membrane phospho• would also lead to a redistribution of cell surface cholesterol, cell sur• face phosphatidylcholine was selectively degraded in fibroblasts, using a phosphatidylcholine-specific from Bacillus cereus. Surpris• ingly, the selective degradation of phosphatidylcholine did not cause a detectable cholesterol translocation in cells (Porn et al., 1993). Under the conditions used, the enzyme degraded about 15% of the total cellular phosphatidylcholine, corre• sponding to 50 DIDol phosphatidylcholine per mg cell protein. The partial degra• dation of phosphatidylcholine left the cells morphologically intact, as evidenced by their exclusion of Trypan blue. Treatment of the same cells with sphingo• myelinase, which resulted in a marked cholesterol redistribution and activation of cholesterol esterification, led to a degradation of about 22 nmol sphingomyelin per mg cell protein. It appears that sphingomyelin depletion alone affects cellular cholesterol distribution, since a selective degradation of plasma membrane phos• phatidylcholine was shown not to have measurable effects on cell cholesterol homeostasis. 284 J. Peter Slotte

5. EFFECTS OF SPHINGOMYELIN ON METABOLISM OF EXOGENOUSLY DERIVED CHOLESTEROL

A substantial fraction of the uptake of exogenously derived cholesterol into cells is mediated by receptor~coupled internalization of LDL particles. This cho• lesterol is eventually transported from lysosomes (the site of LDL degradation) to the plasma membrane compartment. Subsequently, if there is excess free choles• terol in the cell, cholesterol may be transported to the endoplasmic reticulum for esterification by ACAT. Tabas and colleagues have demonstrated that lipoprotein• derived cholesterol mixes rapidly with cellular cholesterol in macrophages, and only move to the substrate pool of ACAT when the cellular cholesterol mass has reached a critical threshold level (Xu and Tabas, 1991). Interestingly, but maybe not surprisingly, it has been shown that this threshold level is regulated by the sphingomyelin content of the cells (Okwu et ai., 1994). These results are entirely compatible with the notion that the sphingomyelin content of cell membranes (mainly plasma membranes) is the major determinant of the solubility of choles• terol in the membranes. Another interesting effect of plasma membrane sphingomyelin on cell cho• lesterol metabolism was observed in the human intestinal CaCo-2 cell line, in which the content of sphingomyelin in the apical membrane was found to regulate cholesterol uptake from bile salt micelles (Chen et al., 1992). A 60% degradation of cell sphingomyelin resulted in a 50% decrease of cholesterol absorption, as well as in a downregulation of endogenous cholesterol biosynthesis. In addition, the basolateral secretion of cholesterol was likewise reduced in sphingomyelin• depleted CaCo-2 cells (Chen et ai., 1992). These authors suggested that the sphingomyelin content of the intestinal cells directly affects cholesterol flux, and that the overall process may be regulated by endogenous sphingomyelinases present in pancreatic juice.

6. CHOLESTEROL-SPHINGOMYELIN INTERACTIONS IN MODEL MEMBRANES

Based on evidence from cell culture systems, it is clear that the cellular homeostasis of cholesterol is directly influenced by sphingomyelin. This fact suggests that these two lipids associate directly in the membrane compartment. What is not clear is why cholesterol prefers to interact with sphingomyelin. An answer to this question is not likely to be gained from experiments at the cellu• lar level, but rather from model membrane studies, in which the level of com• plexity is markedly reduced and the possibility for accurate interpretation of results is higher. Cholesterol-Sphingomyelin Interactions 285

6.1. Affinity of Cholesterol-Sphingomyelin Interaction Compared to Other Phospholipid Systems

Many lines of evidence suggest that the strength or mode of interaction of cholesterol with and sphingomyelins differ. First, the molecular packing properties in cholesteroVsphingomyelin membranes have been suggested to differ from the corresponding cholesterollphosphatidylcholine model membranes (Lund-Katz et aI., 1988), although contradictory results have recently been reported (Smaby et aI., 1994). It has also been shown that water permeability is lower in cholesterollsphingomyelin bilayer membranes than it is in cholesterollphosphatidylcholine systems (Barenholz, 1986), a finding that would be consistent with a more dense lateral packing in cholesteroll sphingomyelin membranes. In addition, it has been established that cholesterol desorption from the membrane into the aqueous phase is markedly reduced in sphingomyelin-containing membranes as compared with phosphatidylcholine membranes of similar acyl chain composition (Lund-Katz et aI., 1988; Bittman, 1993). The slower desorption of cholesterol from sphingomyelin membranes is probably a result of the formation of more attractive van der Waals forces be• tween cholesterol and sphingomyelin. The compressibility modulus has also been reported to be larger in cholesterollsphingomyelin membranes than in cho• lesteroVphosphatidylcholine membranes (Needham and Nunn, 1990). Finally, using cholesterol oxidase as a probe of the strength of interaction between cho• lesterol and sphingomyelin or phosphatidylcholine, it was observed that the oxi• dation susceptibility of cholesterol was much reduced in a sphingomyelin-rich membrane, suggesting a much stronger cholesterol interaction with sphingo• myelin than phosphatidylcholine (Slotte, 1992; Mattjus and Slotte, 1994). Based on all of these results, one can safely postulate that marked differences exist in how cholesterol interacts with sphingomyelin on one hand, and with phos• phatidylcholine on the other.

6.2. Effects of Alterations in the Sphingomyelin Molecule on Cholesterol-Sphingomyelin Interactions

In an effort to examine the molecular explanation(s) for the high affinity of association between cholesterol and sphingomyelin, synthetic analogues of sphingomyelin were prepared in which different functional groups were al• tered systematically (Figure 1). These analogues were incorporated into model membrane systems, together with cholesterol, in order to examine how the functional alterations in the sphingomyelin molecule affected the strength of interaction with cholesterol, as probed with cholesterol oxidase. When the hy• droxy group at carbon 3 of the sphingosine backbone was replaced by a hydro• gen atom (to yield 3-deoxy-sphingomyelin), the oxidation susceptibility of 286 J. Peter Slotte

o

NH

N-palmitoylsphingomyelin

C l7H'5 I C=O I NH 0 CI'H27~oFH2CH2N.Me, o· 3-deoxy-2-N-stearoyl-SPM

C 17H'5 I C=O N-stearoyl-SPM: R = OH I Analogs: R = H, OMe, or OEt o 0 CI3H27~OrCH2CH~.Me, O· 3-deoxy-2-0-stearoyl-SPM

(ester analog of SPM)

FIGURE 1. Schematic molecular structure of N-palmitoylsphingomyelin. See the text for a discussion of the effects of structural alterations in the sphingomyelin molecule on cholesterol-sphingomyelin in• teractions in model membranes. Cholesterol-Sphingomyelin Interactions 287 cholesterol was slightly reduced (Gronberg et at., 1991). However, when other functional groups were attached to the 3-0H group (i.e., a methoxy or ethoxy function) no change was observed in the rate of cholesterol oxidation, sug• gesting that alterations of the 3-0H group has little or no effect on cholesterol-sphingomyelin interaction in model membranes. The length and degree of un saturation (saturated vs mono-unsaturated) of the amide-linked was expectedly shown to affect the strength of cholesterol• sphingomyelin interaction (Slotte, 1992). When the amide-linkage fatty acid was replaced by an ester-linked acyl chain, the strength of interaction between the sphingomyelin and cholesterol decreased markedly (Bittman et aI., 1994). This result implies that the amide-linked fatty acid function in sphingomyelin has a profound effect on the sterol-sphingomyelin interaction. It is possible that the amide-linked fatty acid influences the lateral interaction between sphingomyelin and cholesterol, especially in the polar interface region of the membrane, thereby allowing for a more favorable packing geometry.

6.3. Stoichiometry of the Cholesterol-Sphingomyelin Interaction

The interaction between cholesterol and phosphatidylcholine in model membranes appears to be of an equimolar nature (Lecuyer and Dervichian, 1969; Phillips and Finer, 1974; Collins and Phillips, 1982). When the cholesterol/ phospholipid molar ratio is below 1, clusters of free phospholipid coexist with equimolar cholesterol/phospholipid "complexes" (McLean and Phillips, 1982). On the other hand, if the cholesterol/phospholipid ratio exceeds 1, free choles• terol clusters coexist with cholesterol/phospholipid complexes (Lecuyer and Dervichian, 1969; Collins and Phillips, 1982). When the equimolar stoichiometry is exceeded with respect to cholesterol, free sterol-rich clusters apparently form in the membranes (probably in laterally segregated domains). The observation that phosphatidylcholines can only solubilize an equimolar amount of cholesterol has been demonstrated using several different experimental techniques, both in bilayer and in monolayer membranes. However, when the maximal solubiliz• ing capacity of sphingomyelin was determined in monolayer membranes using cholesterol oxidase as a probe, it was found that one mole of sphingomyelin could solubilize two moles of cholesterol (Slotte, 1992). This 2: 1 cholesterol to sphingomyelin stoichiometry appears to hold, irrespective of the nature of the amide-linked fatty acid function in the sphingomyelin molecule (Slotte, 1992; Slotte, unpublished observations). Even replacement of the amide-linked fatty acid chain by an ester acyl function did not alter the 2: 1 stoichiometry (Bittman et at., 1994). Interestingly, phosphatidylcholines with one or two alkyl-linked chains (either in sn-l or sn-2 positions, or both) also have the capacity to solubi• lize two moles of cholesterol per mole of the alkyl phosphatidylcholine (Mattjus et at., 1996). 288 J. Peter Stolte

7. EFFECTS OF CHOLESTEROL ON PHOSPHOLIPID HOMEOSTASIS

Since the membrane level of sphingomyelin, by affecting the subcellular dis• tribution of cholesterol, can indirectly regulate the rate of cholesterol biosynthesis, one can ask whether changes in plasma membrane cholesterol levels, or changes in cell cholesterol homeostasis in general, also may affect rates of phospholipid biosynthesis. In a preliminary study in which the incorporation of radiolabeled acetate into phosphatidy1choline was examined as a function of cholesterol surface transfer from extracellular vesicles, it was observed that a net flow of cholesterol mass into cultured rat aortic smooth muscle cells led to an increased formation of radiolabeled phosphatidy1choline (Slotte and Lundberg, 1983). In a later study, it was demonstrated that cholesterol loading into macrophages stimulated the biosynthesis of phosphatidy1choline (Shiratori et aI., 1994). In another study with intestinal CaCo-2 cells, it was demonstrated that the regulation of HMG-CoA re• ductase (cholesterol biosynthesis) and palmitoyltransferase (sphingosine synthesis) were independent of each other (Chen et at., 1993). However, it was ob• served that when the membrane mass of either cholesterol or sphingomyelin were altered, parallel changes occurred in the rate of synthesis of these two lipids. In a recent study with Chinese hamster ovary cells, it was noted that 25-hydroxycho• Ie sterol stimulated sphingomyelin synthesis (Ridgway, 1995). This may be of relevance during cholesterol loading of cells, since cells apparently can oxidize cholesterol to 25-and 27 -hydroxycholesterol (Bjorkhem et at., 1994), which in tum may regulate the formation of sphingomyelin. A recent review on phospholipid biosynthesis in eucaryotic cells may also be of interest to the reader (Kent, 1995).

8. CONCLUDING REMARKS

Studies performed so far suggest strongly that plasma membrane sphingo• myelin is the important determinant of cholesterol distribution in the cell. This suggestion does not exclude the possibility that other additional properties of plasma membranes also affect the cellular cholesterol distribution. Although it has not been directly proved that sphingomyelin and cholesterol actually interact with each other in the membrane compartment-such an interaction is suggested based on results obtained from work with both biological and model membranes. An• other question which has yet to be addressed adequately is which species comes first to the plasma membrane-cholesterol or sphingomyelin? And, if sphin• gomyelin is first, is this enough of a reason for cholesterol to also accumulate in this same compartment? Current efforts in many laboratories to elucidate both in• tracellular cholesterol transfer routes and their regulation, and sphingomyelin transfer routes and possible sorting events, is expected to help clarify this picture and allow us to better understand the underlying reasons. Cholesterol-Sphingomyelin Interactions 289

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