Apolipoprotein-mediated cellular lipid release requires replenishment of sphingomyelin in a phosphatidylcholine-specific phospholipase C-dependent manner

When sphingomyelin is digested by sphingomyelinase in the plasma membrane of rat astrocytes, productions of sphingomyelin, diacylglycerol, and phosphatidylcholine are stimulated. D609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed these effects. Similarly, when apolipoprotein A-I removed cellular cholesterol, phosphatidylcholine, and sphingomyelin to generate high density lipoprotein, cholesterol synthesis from acetate subsequently increased, and sphingomyelin synthesis from acetate and serine also increased. D609 inhibited these effects again. D609 also inhibited the cholesterol removal by apoA-I not only from the astrocytes but also from BALB/3T3 and RAW264 cells. D609 decreased cholesterol synthesis, although D609 did not directly inhibit hydroxymethylglutaryl-CoA reductase. ApoA-I-stimulated translocation of newly synthesized cholesterol to cytosol was also decreased by D609. A diacylglycerol analog increased the apoA-I-mediated cholesterol release, whereas ceramide did not influence it. We concluded that removal of cellular sphingomyelin by apolipoproteins is replenished by transfer of phosphorylcholine from phosphatidylcholine to ceramide, and this reaction may limit the removal of cholesterol by apoA-I. This reaction also produces diacylglycerol that potentially triggers subsequent cellular signal cascades and regulates intracellular cholesterol trafficking.     

Modulation of regulatory oxysterol formation and low density lipoprotein suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity by ketoconazole. A role for cytochrome P-450 in the regulation of HMG-CoA reductase in rat intestinal epithelial cells

The effects of ketoconazole, a lanosterol demethylase and cytochrome P450 inhibitor, on the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34, reductase) activity and sterol biosynthesis were studied in rat intestinal epithelial cell cultures (IEC-6). Incubation of cells with 0.15-2 microM ketoconazole resulted in a concentration-dependent inhibition of reductase activity. As the drug concentration approached 15 microM, the reductase activity returned to control values, and at 30 microM ketoconazole, a stimulation of enzyme activity was observed. The drug had no effect on reductase activity in homogenates of IEC-6 cells. Ketoconazole (0.15-30 microM) caused a concentration-dependent inhibition of the incorporation of [3H] mevalonolactone into cholesterol with a concomitant accumulation of radioactivity in methyl sterols; e.g. lanosterol and 24,25-epoxylanosterol. Interestingly, the incorporation of radioactivity into polar sterols showed a biphasic response which was inversely proportional to the biphasic response of reductase activity. Thus, incorporation of [3H]mevalonolactone into polar sterols increased at low concentrations of ketoconazole (0.15-2 microM) and decreased to control values at high concentrations of the drug. Treatment of cells with ketoconazole (30 microM) and [3H]mevalonolactone followed by removal of the drug and radiolabel resulted in an inhibition of reductase activity and a redistribution of radioactivity from lanosterol and 24,25-epoxylanosterol to cholesterol and polar sterols. These results suggested that the inhibition of reductase activity at low concentrations of ketoconazole (less than 2 microM) was due to a formation of regulatory polar sterols generated from the methyl sterols. At high concentrations of ketoconazole (30 microM) where no suppression in reductase activity was observed, the conversion of exogenously added [3H]24(S),25-epoxylanosterol to polar sterols was prevented. Exogenously added 24,25-epoxylanosterol inhibited reductase activity in a dose-dependent fashion, and ketoconazole (30 microM) prevented the inhibition caused by low concentrations of epoxylanosterol. The drug, however, was unable to prevent the dose-dependent suppression of reductase activity by 25-hydroxylanosterol, a reduced form of 24,25-epoxylanosterol. These results indicated that 24,25-epoxylanosterol per se was not an inhibitor of reductase activity but could be metabolized to regulatory polar sterols through a cytochrome P-450 dependent reaction which was sensitive to ketoconazole. Treatment of cells with ketoconazole totally abolished the inhibition of reductase activity by low density lipoprotein (LDL).(ABSTRACT TRUNCATED AT 400 WORDS)     

Sphingolipids and cellular cholesterol homeostasis. Effect of ceramide on cholesterol trafficking and HMG CoA reductase activity

We previously showed that degradation of cellular sphingomyelin (SM) by SMase C results in a greater stimulation of cholesterol translocation to endoplasmic reticulum, compared to its degradation by SMase D. Here we investigated the hypothesis that the effect of SMase C is partly due to the generation of ceramide, rather than due to depletion of SM alone. Inhibition of hydroxymethylglutaryl CoA reductase (HMGCR) activity was used as a measure of cholesterol translocation. Treatment of fibroblasts with SMase C resulted in a 90% inhibition of HMGCR, whereas SMase D treatment inhibited it by 29%. Treatment with exogenous ceramides, or increasing the endogenous ceramide levels also inhibited HMGCR by 60-80%. Phosphorylation of HMGCR was stimulated by SMase C or exogenous ceramide. The effects of ceramide and SMase D were additive, indicating the independent effects of SM depletion and ceramide generation. These results show that ceramide regulates sterol trafficking independent of cellular SM levels.     

Lovastatin reversed the enhanced sphingomyelin caused by 27-hydroxycholesterol in cultured vascular endothelial cells

Statins have pleiotropic properties which are involved in inhibiting the thrombogenic response. In this study, the effects of lovastatin on two phospholipids, phosphatidylcholine and sphingomyelin, were studied in cultured endothelial cells in the presence of an oxysterol, 27-hydroxycholesterol. After the cells were cultured with 50 nM of lovastatin for 60 h, lovastatin was found to decrease the incorporation of [³H]choline into phosphatidylcholine and sphingomyelin, inhibited CTP: phosphocholine cytidylyltransferase (CT) activity without altering the activity of sphingomyelin synthase and neutral sphingomyelinase. And lovastatin was not found to have a direct inhibitive effect on activity of CT. Exogenous mevalonic acid or cholesterol reversed the reduction of cholesterol concentration that was caused by lovastatin, but had no significant effect on the diminished [³H]sphingomyelin by lovastatin. The increase of [³H]sphingomyelin by 27-hydroxycholesterol was not detected in the presence of lovastatin. These findings suggest that (1) lovastatin can reduce sphingomyelin content by means of inhibiting phosphatidylcholine synthesis; and (2) The decrease in sphingomyelin is not related to the diminished cholesterol concentration or mevalonate-derived intermediates. This inhibitive effect of lovastatin on sphingomyelin may benefit cellular calcification caused by sphingomyelin.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols

Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-[2-(diethylamino)-ethoxy]androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis. The epimeric mixture of 24,25-epoxylanosterols could be resolved by high performance liquid chromatography on a wide-pore, non-endcapped, reverse phase column. Both epimers were effective suppressors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity of IEC-6 cells. The suppressive action of the natural epimer, 24(S),25-epoxylanosterol, but not that of 24(R),25-epoxylanosterol could be completely prevented by ketoconazole. IEC-6 cells could efficiently metabolize biosynthetic 24(S),25-epoxy[3H]anosterol mainly to the known reductase-suppressor 24(S),25-epoxycholesterol. This metabolism was substantially reduced by ketoconazole. These data support the conclusion that 24(S),25-epoxylanosterol per se is not a suppressor of HMG-CoA reductase activity but is a precursor to a regulatory oxysterol(s). It has recently been reported that 25-hydroxycholesterol can occur naturally in cultured cells in amounts sufficient to effect regulation of HMG-CoA reductase (Saucier et al. 1985. J. Biol. Chem. 260: 14571-14579). In order to investigate the biological effects of possible precursors of 25-hydroxycholesterol, we chemically synthesized 25-hydroxylanosterol and 25-hydroxylanostene-3-one. Both oxylanosterol derivatives suppressed cellular sterol synthesis at the level of HMG-CoA reductase. U18666A had the unusual effect of potentiating the inhibitory effect of 25-hydroxylanostene-3-one but did not influence the effect of other oxylanosterols. All the oxylanosterols, with the exception of 25-hydroxylanostene-3-one, enhanced intracellular esterification of cholesterol. The foregoing observations support consideration of oxylanosterols as playing an important role in the biological formation of regulatory oxysterols that modulate sterol biosynthesis at the level of HMG-CoA reductase.     

Differential regulation of low density lipoprotein suppression of HMG-CoA reductase activity in cultured cells by inhibitors of cholesterol biosynthesis

Treatment of rat intestinal epithelial cells (IEC-6 cells) with lanosterol 14 alpha-demethylase inhibitors, ketoconazole and miconazole, had similar effects on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and cholesterol biosynthesis but the drugs differed in their ability to prevent the low density lipoprotein (LDL) suppression of reductase activity. Miconazole, at concentrations that inhibited the metabolism of lanosterol and epoxylanosterol to the same degree as ketoconazole, did not prevent low density lipoprotein action on reductase activity, whereas ketoconazole totally abolished the low density lipoprotein action on reductase activity. Both drugs caused: 1) a biphasic response in reductase activity such that at low concentrations (less than 2 microM) reductase activity was inhibited and at high concentrations (greater than 5 microM) the activity returned to control or higher than control levels; 2) an inhibition of metabolism of lanosterol to cholesterol, and 24(S), 25-epoxylanosterol to 24(S), 25-epoxycholesterol. Neither drug prevented suppression of reductase activity by 25-hydroxylanosterol, 25-hydroxycholesterol, or mevalonolactone added to the medium. Each drug increased the binding, uptake, and degradation of 125I-labeled LDL and inhibited the re-esterification of free cholesterol to cholesteryl oleate and cholesteryl palmitate. The release of free cholesterol from [3H]cholesteryl linoleate LDL could not account for the differential effect of ketoconazole and miconazole on the prevention of low density lipoprotein suppression of reductase activity. The differential effect of the drugs on low density lipoprotein suppression of reductase activity was not unique to IEC-6 cells, but was also observed in several cell lines of different tissue origin such as human skin fibroblast cells (GM-43), human hepatoblastoma cells (HepG2), and Chinese hamster ovary cells (wild type, K-1; 4 alpha-methyl sterol oxidase mutant, 215). These observations suggest that the suppressive action of low density lipoprotein on reductase activity 1) does not require the de novo synthesis of cholesterol, or 24(S), 25-epoxysterols; 2) is not mediated via the same mechanism as that of mevalonolactone; and 3) does not involve cholesteryl reesterification. Ketoconazole blocks a site in the process of LDL suppression of reductase activity that is not affected by miconazole.     

Catabolism of Exogenous and Endogenous Sphingomyelin and Phosphatidylcholine by Homogenates and Subcellular Fractions of Cultured Neuroblastoma Cells. Effects of Anesthetics

Cultured murine neuroblastoma cells contain a neutral, Mg2+-stimulated sphingomyelinase and an alkaline phosphatidylcholine-hydrolyzing activity that are enriched in the plasma membrane fraction. The reaction products of sphingomyelin catabolism are phosphocholine and ceramide and those of phosphatidylcholine, glycerophosphocholine and fatty acid. These reactions were studied with endogenous as well as exogenous liposomal substrates. With both exogenous and endogenous substrates, the sphingomyelinase activity was stimulated two- to threefold by Mg2+ and a further three- to fourfold by volatile anesthetic agents. Stimulation was concentration-dependent and corresponded to anesthetic potency: methoxyflurane greater than halothane greater than enflurane. Greater than 80% of the plasma membrane sphingomyelin was hydrolyzed within 2 h in the presence of Mg2+ and anesthetic. In contrast, the activity with exogenous and endogenous phosphatidylcholine was unaffected by Mg2+ or Ca2+ and was markedly inhibited (50-80%) by anesthetic agents. The degree of inhibition was concentration-dependent and corresponded to anesthetic potency. The quantitative importance of choline-containing lipids in cell membranes, the relatively exclusive localization of the neutral Mg2+-stimulated sphingomyelinase in cells of neural origin, the totally different type of hydrolytic attack on phosphatidylcholine, and the reciprocal effects of anesthetics on the hydrolysis of these two lipids strongly suggest important roles for these activities in cell membranes in general and in the neuron in particular.     

Compartmentalization of cholesterol metabolism and cellular growth in cultured intestinal crypt cells

Growth of rat intestinal crypt derived cells IEC-6 ceased when the key enzyme of cholesterol synthesis, hydroxymethylglutaryl-CoA reductase, was blocked by the competitive inhibitor mevinolin. This effect was reversed by the addition of mevalonolactone. LDL suppressed reductase activity as well as cholesterol synthesis from [14C]octanoate and stimulated acyl-CoA cholesterol acyltransferase, but failed to support cell growth despite rapid receptor mediated degradation even in the presence of low mevalonolactone concentrations. Inhibition of cholesterol esterification by Sandoz-Compound 58-035 enhanced cell growth in the presence of mevinolin, but did not promote proliferation in the additional presence of low-density lipoproteins. HDL3 but not HDL2 or tetranitromethane-modified HDL3 totally reversed the mevinolin induced inhibition of cell growth. This rescue by HDL3 was overcome by an increased dose of mevinolin. HDL3 derepressed reductase, stimulated cholesterol synthesis and reduced cholesterol esterification, but did not reverse the cholesterol synthesis inhibition by mevinolin. It is concluded that IEC-6 cells preferentially use endogenously synthesized cholesterol for membrane formation rather than low-density lipoprotein cholesterol. High-density lipoproteins appear to normalize cell growth in the presence of mevinolin by inhibition of cholesterol esterification and probably by inducing the formation of non sterol products of mevalonate.     

Regulation of sterol biosynthesis and of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in cultured cells by progesterone

Treatment of rat intestinal epithelial cells in culture (IEC-6) with progesterone (10 micrograms/ml) caused a strong inhibition of cholesterol biosynthesis as indicated by a decreased incorporation of radiolabel from [3H]acetate. This inhibition was accompanied by an accumulation of radioactivity in an intermediate which coeluted with authentic desmosterol upon high performance liquid chromatography (HPLC). In addition, treatment of cells with progesterone caused lesser accumulation of radiolabel in products with retention times (RT) of 7.9 and 13.5 min on reverse-phase HPLC. The RT-13.5 compound was tentatively identified as cholesta-5,7,24-trien-3 beta-ol based on its relative retention and on its conversion to cholesterol upon incubation with untreated cells. The RT-7.9 compound was identified as 24 (S),25-epoxycholesterol (S-EC) based on its coelution with authentic S-EC and by its conversion to 25-hydroxycholesterol upon reduction with LiAlH4. Incubation of IEC-6 cells with chemically prepared S-EC resulted in dose-dependent suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity within 6 h (I50 = 0.3 microM). Pretreatment of cells with progesterone prevented this suppressive effect. No suppression of reductase activity was observed in progesterone-treated cells in spite of obvious accumulation of S-EC in amounts sufficient to effect regulation; instead, a 2-3-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity occurred within a 24-h period. Following the removal of progesterone from the culture medium, reductase activity declined rapidly over the next 6 h. However, IEC-6 cells could not metabolize S-EC, derived either endogenously or exogenously, during a similar time frame; nor did progesterone affect the uptake of exogenous S-EC by IEC-6 cells. These results show that although progesterone treatment of cultured cells promotes the synthesis of a natural oxysterol suppressor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the continued presence of progesterone prevents the regulatory action of S-EC. The unique nature of this interference is high-lighted by the observation that progesterone could not prevent the suppression of reductase activity by either 25-hydroxycholesterol or mevalonolactone.     

Resynthesis of sphingomyelin from plasma-membrane phosphatidylcholine in BHK cells treated with Staphylococcus aureus sphingomyelinase.

About 60-65% of the total sphingomyelin in intact BHK cells is in a readily accessible pool which is rapidly degraded by Staphylococcus aureus sphingomyelinase. No more sphingomyelin is broken down in cells which have been fixed with glutaraldehyde or lysed with streptolysin O, suggesting that all the sphingomyelin which is available to the enzyme is on the cell surface. The inaccessible pool of sphingomyelin does not equilibrate with the plasma-membrane pool, even after prolonged incubation. Experiments using [3H]-choline show that much more phosphocholine is released from the intact cells treated with sphingomyelinase than can be accounted for by breakdown of the original cell-surface pool of sphingomyelin; the excess appears to be a consequence of the breakdown of sphingomyelin newly resynthesized at the expense of a pool of phosphatidylcholine which represents about 8% of total cell phosphatidylcholine and may reside in the plasma membrane. This would be consistent with resynthesis of cell-surface sphingomyelin by the phosphatidylcholine: ceramide phosphocholinetransferase pathway, which has previously been shown to be localized in the plasma membrane. However, in [3H]palmitate-labelled cells there appeared to be no accumulation of the diacylglycerol expected to be produced by this reaction, and no enhanced synthesis of phosphatidate or phosphatidylinositol; instead there was an increased synthesis of triacylglycerol. A similar increase in labelling of triacylglycerol was seen in enzyme-treated cells where the sphingomyelinase was subsequently removed, allowing resynthesis of sphingomyelin which occurred at a rate of about 25% of total sphingomyelin/h. Treatment of BHK cells with sphingomyelinase caused no change in the rates of fluid-phase endocytosis or exocytosis as measured with [3H]inulin.     

Sphingomyelin modulates capacitation of human sperm in vitro

Ejaculated mammalian sperm must mature (capacitate) before they can undergo acrosomal exocytosis and fertilize an egg. Loss of sperm sterols is an early step in capacitation. Because sphingomyelin slows cholesterol efflux from other cells, the role of sphingomyelin in capacitation was tested. Human sperm were exposed to sphingomyelinase and then incubated for as long as 24 h. The ability of sperm to acrosome-react in response to progesterone was tested to measure capacitation. Sphingomyelinase-treated sperm became responsive to progesterone approximately 10 h earlier than control sperm. Sphingomyelinase also increased spontaneous acrosomal exocytosis. The effects of sphingomyelinase were accompanied by accelerated losses of the inhibitory sterols, cholesterol and desmosterol. To test whether sphingomyelinase-generated ceramide might promote capacitation, sperm were incubated for 8 h with the cell-permeable ceramide N:-hexanoylsphingosine (25 microM) or with solvent. Ceramide increased the incidence of progesterone-responsive sperm and, at later times, spontaneously reacted sperm. N:-Hexanoylsphinganine, an inactive control ceramide, had no effect. These results suggest that sphingomyelin in the sperm influences the rate of capacitation by slowing the loss of sterols, and that exogenous sphingomyelinase accelerates capacitation by speeding the loss of sterols and by generating ceramide.     

Trifluoperazine stimulates the coordinate degradation of sphingomyelin and phosphatidylcholine in GH3 pituitary cells

Prior studies demonstrated that 1,2-diacylglycerols stimulated degradation of the choline-containing phospholipids, phosphatidylcholine and sphingomyelin, in GH3 pituitary cells by a phospholipase A2 and a sphingomyelinase, respectively (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762). The present studies demonstrate that the phenothiazine trifluoperazine also stimulates degradation of these phospholipids. Trifluoperazine (25 microM) reduced phosphatidylcholine and sphingomyelin levels to 81 and 58% of control, respectively, after 30 min in cells labeled for 48 h with [3H] choline. Choline-containing metabolites were released specifically into the cytosolic fraction. The level of cytosolic phosphocholine, but not choline or CDP-choline, increased to 150% of control. These events were not mediated by inhibition of phosphatidylcholine synthesis. The level of 1,2-diacylglycerols, but not lysophosphatidylcholine or glycerol-3-phosphocholine, also increased. These data are most consistent with phosphatidylcholine degradation via a phospholipase C. Trifluoperazine-stimulated sphingomyelin degradation was accompanied by quantitative generation of ceramides consistent with activation of a sphingomyelinase. In contrast to trifluoperazine, choline-containing metabolites were released into the medium during stimulation by the 1,2-diacylglycerol 1,2-dioctanoyl-glycerol. Although both trifluoperazine and 1,2-dioctanoylglycerol increased ceramide levels, only 1,2-dioctanoylglycerol increased the sphingoid base level from 24 to 43 pmol/10(6) cells. Hence, trifluoperazine appears to deplete an intracellular pool of phosphatidylcholine and sphingomyelin by a different mechanism than 1,2-diacylglycerols. This is the first report of phenothiazine-induced degradation of choline-containing phospholipids.     

Oxysterol-binding protein and vesicle-associated membrane protein-associated protein are required for sterol-dependent activation of the ceramide transport protein

Sphingomyelin (SM) and cholesterol are coregulated metabolically and associate physically in membrane microdomains involved in cargo sorting and signaling. One mechanism for regulation of this metabolic interface involves oxysterol binding protein (OSBP) via high-affinity binding to oxysterol regulators of cholesterol homeostasis and activation of SM synthesis at the Golgi apparatus. Here, we show that OSBP regulation of SM synthesis involves the endoplasmic reticulum (ER)-to-Golgi ceramide transport protein (CERT). RNA interference (RNAi) experiments in Chinese hamster ovary (CHO)-K1 cells revealed that OSBP and vesicle-associated membrane protein-associated protein (VAP) were required for stimulation of CERT-dependent ceramide transport and SM synthesis by 25-hydroxycholesterol and cholesterol depletion in response to cyclodextrin. Additional RNAi experiments in human embryonic kidney 293 cells supported OSBP involvement in oxysterol-activated SM synthesis and also revealed a role for OSBP in basal SM synthesis. Activation of ER-to-Golgi ceramide transport in CHO-K1 cells required interaction of OSBP with the ER and Golgi apparatus, OSBP-dependent Golgi translocation of CERT, and enhanced CERT-VAP interaction. Regulation of CERT by OSBP, sterols, and VAP reveals a novel mechanism for integrating sterol regulatory signals with ceramide transport and SM synthesis in the Golgi apparatus.     

beta-Sitosterol stimulates ceramide metabolism in differentiated Caco2 cells

Previous studies from our laboratory on tumor cells suggest that phytosterols stimulate ceramide production, which was associated with cell growth inhibition and stimulation of apoptosis. The objective of the present study was to examine the effect of phytosterols on ceramide metabolism in small intestinal cells that represent the first cells in contact with dietary phytosterols. Caco(2) cells, an accepted model for human intestinal epithelial cells, were used in this study. Ceramide and ceramide-containing lipids were examined by labeling the ceramide pool with (3)H-serine. Cells were supplemented with 16 microM of sterols (cholesterol, beta-sitosterol or campesterol) for 16 days postconfluence and continued to differentiate. Of the two phytosterols, beta-sitosterol, but not campesterol, induced more than double the serine labeling when compared with cholesterol. This increase was uniform in sphingomyelin (SM), ceramide and sphingosine labeling. Sterols had no effect on SM concentration in the cells. In addition, sterol had no effect on the activity of SM synthase or sphingomyelinases. There was an inhibition of ceramidases with campesterol supplementation. These data suggest that the observed increases in SM and sphingosine labeling were due to an increase in ceramide turnover. The increase in ceramide turnover with beta-sitosterol supplementation was not associated with growth inhibition but was with increases in ceramide glycosylation products such as cerebrosides and gangliosides. It was concluded that beta-sitosterol has no effect on differential Caco(2), a model of normal small intestinal cells. The increase in the glycosylated ceramide products may offer a means to protect the cells from the harmful effect of ceramide by excreting them with lipoproteins.     

Sterol synthesis is up-regulated in cholesterol-loaded pigeon macrophages during induction of cholesterol efflux.

The extent to which cholesterol synthesis is modulated in macrophage foam cells by changes in cholesterol influx and efflux was determined using thioglycollate-elicited peritoneal macrophages from normal and cholesterol-fed White Carneau (WC) and Show Racer (SR) pigeons. In peritoneal macrophages from normocholesterolemic pigeons, sterol synthesis from [(14)C]-acetate was down-regulated by more than 90% following incubation in vitro with beta-VLDL. Sterol synthesis was increased when the cellular free cholesterol concentration was decreased in response to stimulation of cholesterol efflux with apoHDL/phosphatidylcholine vesicles and cyclodextrin. Peritoneal macrophages isolated from hypercholesterolemic pigeons were loaded with cholesterol to levels similar to foam cells from atherosclerotic plaques (375-614 microg/mg cell protein), and had an extremely low rate of sterol synthesis. When cholesterol efflux was stimulated in these cells, sterol synthesis increased 8 to 10-fold, even though the cells remained grossly loaded with cholesterol. Cholesterol efflux also stimulated HMG-CoA reductase activity and LDL receptor expression. This suggests that only a small portion of the total cholesterol pool in macrophage foam cells was responsible for regulation of sterol synthesis, and that cholesterol generated by hydrolysis of cholesteryl esters was directed away from the regulatory pool by efflux from the cells. When the increase in sterol synthesis was blocked with the HMG-CoA reductase inhibitor mevinolin, there was no difference in the cholesterol content of the cells, or in the mass efflux of cholesterol into the culture medium.Thus, under these conditions, the increase in cholesterol synthesis during stimulation of cholesterol efflux does not appear to contribute significantly to the mass of cholesterol in these macrophage foam cells. Whether a similar situation exists in vivo is unknown.     

A lipid analogue that inhibits sphingomyelin hydrolysis and synthesis, increases ceramide, and leads to cell death

We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 microM led to an increase in cellular ceramide and cell death. Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells.     

The role of sphingomyelin in regulating phase coexistence in complex lipid model membranes: competition between ceramide and cholesterol

The structure, thermotropic phase behavior, dynamic motion and order parameters of bilayer dispersions of egg phosphatidylcholine, egg sphingomyelin, egg ceramide and cholesterol have been determined. The coexistence of gel, liquid-ordered and liquid-disordered structure has been determined by peak fitting analysis of synchrotron X-ray powder patterns. Order parameters and extent of distribution of 16-doxyl-stearic acid spin probe between ordered and disordered environments has been estimated by ESR spectral simulation methods. The presence of ceramide in proportions up to 20 mol% in phosphatidylcholine is characterized by gel-fluid phase coexistence at temperatures up to 46 degrees C depending on the amount of ceramide. Cholesterol tends to destabilize the ceramide-rich domains formed in phosphatidylcholine while sphingomyelin, by formation of stable complexes with ceramide, tends to stabilize these domains. The stability of sphingomyelin-ceramide complexes is evident from the persistence of highly ordered structure probed by ESR spectroscopy and appearance of a sharp wide-angle X-ray reflection at temperatures higher than the gel-fluid transition of ceramide alone in egg phosphatidylcholine bilayers. The competition between ceramide and cholesterol for interaction with sphingomyelin is discussed in terms of control of lipid-mediated signaling pathways by sphingomyelinase and phospholipase A2.     

Sphingomyelin metabolites inhibit sphingomyelin synthase and CTP:phosphocholine cytidylyltransferase

Tissue injury in inflammation involves the release of several cytokines that activate sphingomyelinases and generate ceramide. In the lung, the impaired metabolism of surfactant phosphatidylcholine (PC) accompanies this acute and chronic injury. These effects are long-lived and extend beyond the time frame over which tumor necrosis factor (TNF)-alpha and interleukin-1beta are elevated. In this paper, we demonstrate that in H441 lung cells these two processes, cytokine-induced metabolism of sphingomyelin and the inhibition of PC metabolism, are directly interrelated. First, metabolites of sphingomyelin hydrolysis themselves inhibit key enzymes necessary for restoring homeostasis between sphingomyelin and its metabolites. Ceramide stimulates sphingomyelinases as effectively as TNF-alpha, thereby amplifying the sphingomyelinase activation, and TNF-alpha, ceramide, and sphingosine all inhibit PC:ceramide phosphocholine transferase (sphingomyelin synthase), the enzyme that restores homeostasis between sphingomyelin and ceramide pools. Second, ceramide inhibits PC synthesis, probably because of its effects on CTP:phosphocholine cytidylyltransferase, the rate-limiting enzymatic step in de novo PC synthesis. The data presented here suggest that TNF-alpha may be an inhibitor of phospholipid metabolism in inflammatory tissue injury. These actions may be amplified because of the ability of metabolites of sphingomyelin to inhibit the pathways that should restore the normal ceramide-sphingomyelin homeostasis.     

Functional reconstitution of sphingomyelin synthase in Chinese hamster ovary cell membranes.

Sphingomyelin synthase (phosphatidylcholine:ceramide phosphocholinetransferase) activity in the membranes of Chinese hamster ovary cells was found to be detectable with a fluorescent ceramide analog, containing a short acyl chain, as a substrate. We developed a method for the functional reconstitution of sphingomyelin synthase in detergent-treated membranes. Treatment of membranes with 1.5% octyl glucoside in the absence of exogenous phosphatidylcholine resulted in almost complete loss of sphingomyelin synthase activity, even after removal of the detergent by dialysis. In contrast, membranes treated with the detergent in the presence of exogenous phosphatidylcholine showed partial activity and, after dialysis of this mixture, enzyme activity was restored to almost the same level as the activity in dialyzed intact membranes. The effects of various lipids on enzyme activity in this reconstitution system suggested that L-alpha-phosphatidylcholine was the environmental lipid essential for the functional reconstitution of the enzyme. Furthermore, diacylglycerol was suggested to serve as an inhibitory regulator of sphingomyelin synthesis.