Intracellular transport of cholesterol in type C Niemann-Pick fibroblasts

Biochimica et Biophysica Acta, 1005 (1989) 303-309 Elsevier

303

BBALIP 53232

Intracellular transport of cholesterol in type C Niemann-Pick fibroblasts J. P e t e r Slotte 1, G u n Hedstrt~m i a n d E d w i n L. B i e r m a n

2

i Department of Biochemistry and Pharmacy, ABO A KADEML Turku (Finland) and 2 Division of Metabolism, Endocrinology, and Nutritio., Department of Medicine, University of Washington, Seattle, WA (U.S.A.)

(Received 11 May 1989)

Key words: Niemann-Pick type C fibroblast; lntracellular transport; Cholesterol oxidase; Sphingomyelinase; Low density lipoproteln; Cholesterol esterification

Tile purpose of this study was to determine tile capadty of Niemann-Pick type C (NPC) fibroblasts to transport cholesterol from the cell surface to in~'acel|ular membranes. This is relevant in light of the observations that NPC cells display a sluggish metabolism of LDL-derived cholesterol, a phenomenon which could be explained by a defective intracellular transport of cholesterol. Treatment of NPC cells for 4 h with 0.1 m g / m l of LDL failed to increase the incorporation of [14C|oleic acid into cholesterol [14C]oleate, an observation consistent with previous reports on this cell type (Pentchev et al. (1985) ~roc. Natl. Acad. Sci. USA 82, 8247). Normal fibroblas,~, however, displayed the classical upregulation (6-fold over control) of the endogenous esterification reaction in response to LDL exposure. ]Incubation of normal or NPC fibroblasts with sphingomyelinase (100 m U / m l ; Staphylococcus aureus) led to a rapid and marked increase (9- and 10-fold for normal and NPC fibroblasts, respectively, after 4 h) in the esterification of plasma-membrane-derived [aHlcholesterol suggesting that sphingomyelin degradation forced a net transfer of cholesterol from the cell surface to the endoplasmic reticulum. The similar response in normal and mutant fibroblasts to the degradation of sphingomyelin suggests that plasma membrane cholesterol can be transported into the substrate pool of ACAT to about the same extent in these two cell types. Degradation of cell sphingomyelin in NPC fibroblasts also resulted in the movement of 20-25% of the cellular cholesterol from a cholesterol oxidase susceptible pool into oxidase-resistant pools, implying that a substantial amount of plasma membrane cholesterol was internalized after sphingomyelin degradation. This cholesterol internalization was not accompanied by an increased rate of membrane internalization, as measured by [3H]sucrose uptake. Although NPC cells showed a relative accumulation of unesterified cholesterol and a sluggish esterification of LDL-derived cholesterol when exposed to LDL, these cells responded like normal fibroblasts with regard to their capacity to transport cholesterol from the cell surface into intracellular sites in response to sphingomyelin degradation. It therefore appears that NPC cells, in contrast to the impaired intraceilular movement of |ipoprotein-derived cholesterol, do not display a general impairment of cholesterol transport between the cell surface and the intracellular regulatory pool of cholesterol.

Introduction Type C Niemann-Pick disease in humans is an autosomal-recessive lipid storage disorder. The common

Abbreviations: LPDP, lipoprotein-deficient plasma; LDL, low-density lipoprotein; DMEM, Dulbecco's modified Eagle's medium; Hep~s, N-2-hydroxyethylpiperazine N-2-ethanesulphonic acid; NPC, Niemann-Pick type C; ACAT, acyI-CoA :cholesterol acyltransferase (EC 2.3.1.26). Correspondence: J.P. Slolte, Department of Biochemistry and Pharmacy, A,BO AKADEMI, Porthansgatan 3, SF-20500 Turku, Finland.

characteristics of this disorder include chronic neurological deterioration associated with hepatomegaly, foamy macrophage infiltration of tissues, and a modest accumulation of sphingomyelin and cholesterol in certain tissues [1,2]. Although the metabolic basis for type C Niemann-Pick disease is still unknown, it has been observed that cells from affected individuals display a decreased esterification of exogenously derived cholesterol [2,3]. Since the impaired esterification of exogenously derived cholesterol in NPC cells is not accounted for by a deficiency in the activity of acylCoA:cholesterol acyltransferase (ACAT; [4]), it has been suggested that the lesion in these mutant cells would be an impaired intracellular transport of

0005-2760/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

304 cholesterol from lysosomes to the site of esterification. Consistent with this hypothesis, it has been shown that cultured NPC fibroblasts accumulate unesterified cholesterol in lysosome-like organelles [2]. The objective of this study was to determine whether the general movement of cholesterol from the cell surface to the putative intracellular regulatory pool of cholesterol was impaired in NPC fibroblasts. Mutant cells were treated with sphingomyelinase to induce a redistribution of plasma membrane cholesterol within the cell [5]. The extent of cholesterol movement was deterrained by measuring the appearance of cell-surface-derived [~H]cholesterol in the substrate pool of ACAT, by measuring the regulatory response in cholesterol biosynthesis from the incorporation of [~C]acetatc into [~4CJC27 sterols, and by determining the distribution of cellular cholesterol between the cell surface and intracellular membranes, as probed with the cholesterol ~xidase technique [6],

Expertmenlal Cell culture. Human skin fibroblasts were obtained from skin biopsies of healthy volunteers. Mutant human NPC fibroblasts (GM 0110A) were purchased from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ, U.S.A.). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10~ fetal calf serum. Cells for experiments were seeded in 35 mm diameter cell culture dishes (at about 50000 cells/dish) and were grown to con£,~enc~ or pre-treated prior to experiments as described separateiy in legends to figures. Isolation of LDL Low density lipoproteins ( d ~ 1.019-1.063 g/ml) were prepared from human plasma (EDTA 4 raM) as described previously [7]. The LDL stock solution (about 11-15 mg/ml)was stored under N= in the dark at 4°C until used within 2 weeks of preparation. Lipoprotein-deficient plasma (LPDP) was prepared at a density of 1.21 g/ml by ultracentrifugation, and was dialysed extensively against phosphate. buffered saline (pH 7.4)/1 mM EDTA before use.

Effects of LDL on [l~C]oleic acid incorporation into cholesteryl [l~C]oleate. Cells to be used for [14C]oleic acid incorporation into cholesterol esters were kept for 48 h in a DMEM supplemented with 10~ LPDP. Cells were then exposcc! to LDL for 4 h 0.0 ml serum.fr¢~ DMEM with 0.1 mg LDL). During the last hour of the 4 h period, 1 ~Ci [14C]oleic acid (20/~M) was added to the dishes. Control cells were incubated in serum-free DMEM without I DL.

Effects of sphingomyeh'nase treatment on [14C]acetate incorporation into C, 7 sterols. Confluent normal or NPC fibroblasts were kept for 48 h in 10~ LPDP prior to the experiments. Cells were exposca, for 4 h to sphingomyefinase (100 mU/ml) in serum-free DMEM. Control

cells received solvent alone. During the last hour of the 4 h period, 0.5 #Ci of [~4C]acetate (10/~M) was added to each dish and the incorporation of [~4C]acetate into [14C]C2~ sterol was determined from the neutral lipid extract.

Effects of sphingomyelinase treatment of the esterification of plasma-membrane.derived [~H]cholesterol. To label the cellular membranes with [3H]cholesterol, confluent normal and NPC fibroblasts were incubated for 2 days in growth medium containing 10% fetal calf serum and [3H]cholesterol (0.5-1.0 ~Ci/ml). The cehs were further incubated for 24 h in serum-free DMEM prior to the experiments. With this labeling procedure, cells contained less than 2~ esterified [3H]cholesterol at the start of the experiment. Cells were then exposed to 100 mU/ml oi' sphingomyelinase (in serum-free DMEM) and the formation of cellular [3H]cholesteryi esters was determined from the neutral lipid extract.

Effects of sphingomyelinase treatment on the distribution of [~H]cholesteroi between cholesterolooxidase.susceptible and .resistant pools. Normal and NPC fibroblasts, labeled with [3H]cholesterol, were exposed to 100 mU/ml of sphingomyelinase for up to 4 h. At indicated ~ time intervals, cells were washed, fixed with 1~ glutaraldehyde in phosphate-buffered saline (pH 7.4), and 0xposed to 1 U cholesterol oxidase and 100 mU sphingomyelinase as described previously [6]. The ratio of [3H]cholesterol and [3H]cholestenone was determined from the neutral lipid extract.

Effects of sphingomyelinase treatment on [3H]sucrose uptake in NPC fibroblasts. The apparent rate of fluidphase pin~:ytosis was determined in control and sphingomyelinase treated NPC fibroblasts from the time-dependent uptake of [3H]sucrose. Confluent cells were incubated at 37°C in 1 ml serum-free medium (containing 10/~M sucrose and 5/~Ci [ 3H]sucrose tracer) with or without 100 mU sphingomyelinase. Uptake of [3H]sucrose to cells incubated at 4°C for 2 h were taken to represent pinocytosis-independent cell adsorption of the tracer, aad these counts were subtracted from the counts obt'~ined at 37°C. An aliquot of the incubation medium was counted to give the total counts and the specific activity per volume unit. Values are given as/~! pinocytosis per mg cell protein. Assay procedures. Cell lipids were extracted with hexane/isopropanol (3/2, v/v) as described [6]. Labeled sterols from cell extracts were isolated on normal phase thin-layer chromatography (Kodak silica-gel plates) in hexane/diethy! ether/acetic acid (130:30:1.5, v/v) and detected by 12 staining. Spots for [3H]- or [14C]cholesterol (R v 0.15-0.20), [3H]cholestenone (R F 0.25-35), and [3H]cholesteryl esters (or cholesterol [14C]oleate) (Rl~ 0.91-0.95) were identified from standards run in parallel. The appropriate spots were marked, the 12 stain was removed and the spots cut into scintillation vials. The radioactivity was counted with 3

305 ml of a xylene-based scintillation cocktail in a LK~ RackBeta liquid scintillation counter, The mass of cellular free and esterified cholesterol was determined from extracts of the appropriate spots on the thin-layer chromatogram by the cholesterol oxid~:se method [8]. Cell proteins in dishes after lipid extraction were digested into 1.0 ml 0.1 M NaOH (60 min) and the protein concentration in an aliquot of the hydrolysate was determined according to Lowry et al.

[91. Results Accumulation and esterification of cholesterol in cells exposed to low-density lipoproteins One of the characteristic features of type C Niemann-Pick cells is the sluggish esterification of lipoprotein-derived cholesterol and the relative accumulation of unesterified cholesterol in these cells [2,3]. To confirm this characteristic feature, we incubated normal and type C Niemann-Pick fibroblasts for 48 h in serum-free DMEM with either 10% lipoprotein-deficient plasma (LPDP) or 0.2 m g / m l of LDL and determined the cellular content of sterol mass (Table I). Treatment of normal cells with LDL increased the free cholesterol content by about 13% compared with the LPDP-treated control cells, whereas the free cholesterol content in type C NiemanmPick fibroblasts increased by 104%. The mass of esterified cholesterol increased with LDL treatment by 290% and 341% in normal and type C Niemann-Pick fibroblasts, respectively (Table I). Although both cell types increased their content of esterifled cholesterol to about the same extent, type C Niemann-Pick fibroblasts accumulated markedly more unesterified cholesterol compared with normal cells. To determine the acute capacity of normal and mutant cells to up-regulate the acyl-CoA:cholesterol

TABLE !

Cell cholesterol mass in normal and type C Niemann-Pick fibroblasts Cells in 35 mm diameter dishes were grown to confluency in DMEM with 10% serum. When confluent (after about 7-9 days) the cells were washed once with 2 ml PBS, switched to serum-free DMEM containing either 10% LPDP or 0.2 mg/ml LDL and incubated for 48 h. After extensive washing with PBS (5 x 2 ml) cellular neutral lipids were extracted and the mass of free and esterified cholesterol was determined. Values are expressed as mean 5: S.D. of triplicale dishes. Cell type

Pretreatment

Cell cholesterol mass (nmol/mg cell protein) free

esterified

HSF

LPDP 48 h LDL 48 h

78.0+ 2.9 88.1 + 9.6

2.1 5:0.6 8.2 + 1.1

NP-C

LPDP 48 h LDL48 h

73.2 + 4.7 149.2+11.9

5.6 + 1.4 24.7+3.7

TABLE 11

Effect of LDL on [t4C]olew acid incorporation into cholesterol [14C]oleate in normal and type C Niemann-Ptck fibroblasts Cells were grown to confluency in DMEM with 19% fetal calf serum. When confluent, the cells were wasl:~d with PBS ( 1 × 2 ml), and incubated for 48 h in DMEM with 10% LPDP. Cells were then washed once with PBS and incubated in serum-free DMEM with or without 0.1 mg/ml of LDL protein for 4 h. Incorporation of [~4C]ole~c acid into cholesterol-[~4C]oleate was determined using a 1 h pulse of cells with 1 p C l / m l of tracer at the end of the 4 h incubation. Values are expressed as mean 5-S.D. of five dishes from two separate experiments. Cell type

Treatment

Cholesteryl [ la C]oleate (cpm/mg protein)

HSF

none I D L 0.1 mg/ml, 4 h

388:t: 15 (100%) 22525:101 (580%)

NP-C

none LDL0.1 mg/ml, 4 h

70~ ± 40 (100%) 801:t: 66(113%)

acyltransf~rase re-lvtion, cells were incubated for 4 h with 0.1 mg/ml of LDL protein, and the incorporatien of [14C]oleic acid into cholesterol [14C]oleate was determined. Treatment of normal fibroblasts witb LDL led to the classic upregulation of the apparent ACAT activity (a 6-fold increase compared to control cells: Fable II). However, no such increase in cholesterol [~4C]oleate formation was observed in type C NiemannPick fibroblasts expc'sed to LDL (Table II). Both the predominant accumulation of unesterified cholesterol in fype C Niemann-Pick fibroblasts and the sluggEh up,'egulation of ACAT following exposure to LDL confirm previous reports on this cell type and support the hypothesis that the intracellular transport of ch,~lesterol to the site of esterification is impaired [2].

Effects of sphingomyelinase-treatment on estcrification of plasma-membrane-derived [ ~H]chotesterol To examine whether the general intracellular movement of cholesterol from the cell surface to the endoplasmic reticulum was impaired in mutant cells, redistribution of plasma membrane cholesterol was induced by sphingomyelinase treatment. Degradation of sphingomyelin in normal fibroblasts is known to cause a rapid movement of plasma membrane cholesterol from the cell surface into the cells, resulting in a dramatic increase of ACAT activity and in a decreased biosynthesis of cholesteroi i5,6]. Incubation of [ 3H]cholesterol-labeled normal fibroblasts for 4 h with serum-free DMEM did not increase the cellular amount of [3H]cholesteryi esters above the initial level (about 1%) measured at the start of the incubation (Fig. 1). Exposure of cells to sphingomyelinase (100 mU/ml) resulted, however, in a marked stimulation of the formation of [3 H]cholesteryl esters (Fig. 1), consistent with previous observations from our group

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Fil. I. Effect of sphiogomyelinase treatment on the esterification of plasma.membrane.derived [SH]cholesterol in normal human fibroblasts. Confluent ~lls in 35 mm diameter dishes were incubated for 48 h in DMEM with 10~ fetal calf serum and 0.5-1.0/tCi [ 3H]cholesterol per dish. Cells were then switched to a serum.free DMEM for another 24 h prior to the experiments, After one wash with PBS, 1,0 ml serum-free medium was added to each dish together with 0.1 U sphinsomyelinase. Control cells received no sphingomyelinase. Cells were then incubated on a 37 ° C water-bath for different periods of time (up to 4 h) at the end of each time interval, the medium was removed, and the cells rinsed with chilled PBS and stored frozen ( - 20 o C) until lipid analysis. The esterification of plasma membrane derived ! s H]cholesterol was determined from the neutral lipid extract. Values are expressed as % [3H]cholesteryl ester over total cell [~H]sterol and are given as meand:S.D, of triplicate dishes from two separate experiments.

[5,6]. The amount of [SH]cholesteryl esters was about 0.$~$ in NPC fibroblasts at the end of the 48 h labeling period. The amount of cell.associated [3H]cholesteryl esters did not increase above the starting level in control cells during the 4 h experiment. However, exposure of NPC fibroblasts to 100 mU/ml of sphingomyelinase also resulted in a rapid and dramatic stimulation of the [SHJcholesteryl ester formation. The onset of the increase in the formation of [3H]cholesteryi esters in response to sphingomyelinase treatment was slightly

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INCUBATIONTIME(hr) Fig. 2. Effect of sphingomyelinase treatment on the esterification of plasma-membrane.derived [3H]cholesterol in Niemann-Pick type C fibroblasts. Confluent NPC cells in 35 nun diameter dishes were treated exactly as described for normal human fibroblasts in the legend to Fig. 1. Values are expressed as % [3H]cholesteryl ester formed and are given as mean + S.D. of tripficate dishes from two separate experiments.

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[t4C]C27 sterol synthesis (cpm/mg cell protein) control

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2002:!:200 893:1: 89

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delayed in NPC cells (Fig. 2) compared with normal fibroblasts (Fig. 1). The rate of ['~H]cholesteryl ester increase was, however, identical in both cell types (a linear regression calculation of the data between the 1st and the 4th h for each curve gives an average increase in [3H]cholesteryl ester formation corresponding to 2.02~ per h and 1.99~ per h for normal and NPC fibroblasts, respectively).

Effects of sphingomyelinase treatment on [14C]acetate incorporation into [ t 4C]C2~ sterols In addition to the stimulation of the [3H]cholesteryl ester formation, the degradation of cell sphingomyelin in NPC fibroblasts also led to a down-regulation of the incorporation of [14C]acetate into [~4C]C27 sterols (Table I11). This finding is similar to the previous observation in normal fibroblasts [5], and further strengthens the view that degradation of cell sphingomyelin resulted in a net flux of plasma membrane cholesterol into the cell, where some of it entered the putative intracellular regulatory pool of cholesterol. Effects of sphingomyelinase treatment on the distribution of cell cholesterol between cholesterol oxidase susceptible and resistant pools In a previous report [6], we showed that treatment of normal fibroblasts with sphingomyelinase within 90 rain led to the transport of about 30% of cell unesterified cholesterol from a cholesterol-oxidase-susceptible pool (i.e., the plasma membrane) to a resistant pool (i.e., undefined intraceilular compartments). In this study we treated [3H]cho|esterol-labeled NPC fibroblasts with 100 mU/ml sphingomyelinase and determined the distribution of cell [3H]cholesterol between cholesterol oxidase susceptible and resistant pools at different time intervals after the onset of sphingomyelinase exposure. We found that at time zero, over 90~ of the cell [~H]cholesterol was available for oxidation (Fig. 3).

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Fig. 3. Distributi6n of cellular [3H]cholesterol between cholesteroloxidase-susceptible and -resistant pools after treatment with sphingomyelinase. Confluent NPC cells in 35 mm diameter dishes were labeled with [3Hlcholesterol as described previously. Labeled cells were kept in serum-free DMEM for 24 h prior to the experiments. They were then rinsed once with PBS and 1.0 mi serum-free medium containing 0.1 U / m l of splfingomyelinase was added to each dish. At time intervals (0 to 4 h), cells were rinsed with PBS/EDTA (5 mM), fixed with glutaraldehyde and treated with cholesterol oxidase. The per cent oxidation is defined as [[ 3Hlcholestonone/([ 3Hlcholestenone +[all]cholesterol)].100. Values are averages from triplicate dishes fron~ two separate experiments + S.D.

With time, however, the oxidase susceptible fraction of cell [3H]eholesterol decreased, and within 120 min it was only about 75% of the total cell [3H]cholesterol (Fig. 2). It therefore appears that degradation of cell sphingomyelin forced 20-25% of the celi-:urf,::,cv cholesterol ~o be transported into the cell, w h e r e , , resistant to the action of cholesterol oxidase.

Effects of sphingomyelinase treatment on the apparent rate of fluid-phase pinocytosis in NPC fibroblasts The transport of about 25% of the cell cholesterol from the cell surface into intracellular compartments in 1.00

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Fig. 4. Effect of sphingomyelinase treatment on [3H]sucrose uptake. Confluent cells were incubated at 37 ° C in 1 mi serum-free medium (containing 10 FM sucrose and 5 FCi [3Hlsucrose tracer) with or without 100 mU sphingomyelinase. Uptake of [3H]sucrose to cells incubated at 4 ° C for 2 h were taken to represent pinocytosis-independent cell adsorption of the tracer, and these counts were subtracted from the counts obtained at 37 ° C. Values are averages+ S.D. of a representative experiment with triplicate dishes for each time point.

sphingomyelinase-treated NPC fibroblasts could have been mediated by a sphJngomyelinase induced microvesiculation of the plasma membrane accompanied by an increased fluid-phase pinocytosis activity [10]. To test for this possibility, we determined the effects of splfingomyelinase treatment of NPC fibroblasts on the uptake of [3H]sucrose. The rate of fluid-phase pinocytosis at 37°C in confluent NPC fibroblasts was about 0.18 F l / h and mg cell protein, as determined from temperature-corrected [3H]sucrose uptake (Fig. 4). Treatment of NPC fibroblasts with 100 m U / m l of sphingomyelinase decreased slightly the amount of [3H]sucrose uptake, to about 0.15 #l/h and mg cell protein. The uptake of [3H]sucrose by sphingomyelinase-treated cells at 4 h was not significantly different from the uptake in control cells at 4 h ( P = 0.07), as shown in Fig. 4. Discussion The results of this study confirm previously published reports [2-4,11] that NPC fibroblasts display defective metabolism of LDL-derived cholesterol. When incubated with LDL, these mutant cells accumulated proportionately more free cholesterol than normal cells (Table I), although they also gained cholesteryl ester mass. The mutant NPC fibroblasts also failed to increase the incorporation of [14C]oleic acid into cholesterol [14C]oleate in response to a short-term LDL exposure, in marked contrast to the normal fibroblasts used in this study. The main objective of this study was to test whether the movement of cholesterol from plasma membranes into the cells also is retarded in type C Niemann-Pick fibroblasts. Sphingomyelinase can be used to degrade sphingomyelin at the cell surface, which in turn leads to a net flow of cholesterol from the cell surface into the cell, some of which is eventually esterified by ACAT [5]. In contrast to the failure of the NPC fibroblasts to respond to a short-term LDL exposure with increased ACAT activity, these cells showed a rapid and marked stimulation of the esterification of plasma membranederived [3H]cholesterol, when treated with sphingomyelinase. Although the esterification response in NPC fibroblasts was slightly delayed compared to the corresponding response in normal fibroblasts, the rate of [3H]cholesteryl ester formation was similar in normal and NPC fibroblasts. Thus it is evident that degradation of sphingomyelin in both normal and NPC fibroblasts led to a dramatic flow of cell surface cholesterol into the substrate pool of ACAT (in the endoplasmic reticulum). This sphingomyelinase-induced flow of cholesterol also led to a reduction of sterol synthesis as reflected by the decreased incorporation of [14C]acetate into [14C]C27 sterols. Such a down-regulation of [14C]precursor incor-

308 poration into [t4C]sterols was not observed when NPC fibroblasts were exposed to LDL cholesterol [11]. Cholesterol oxidase has been used as a probe for assessing the distribution of cholesterol between the cell surface (oxidase susceptible) and intracellul~r (oxidase resistant) compartments [6,7,12-14]. In a previous study [6] we showed that sphingomyelin limits the susceptibility of cholesterol for oxidation by cholesterol oxidase. Similar findings on related systems have been reported previously [15,16]. The experiments with NPC fibroblasts in this study showed that more than 90~ of the cellular unesterified cholesterol was susceptible for oxidation, and hence was probably located at the cell surface. However, in cells exposed for 4 h to sphingomyelinase only about 75% of the cell cholesterol was oxidi~ble, suggesting that 20-25% of the cell surface cholesterol had been translocated into the cell (no cholesterol was lost due to efflux). Sphingomyelinase treatment of red blood cells is reported to result in a fairly dramatic endovesiculation of the membrane, with about 25~ of the cell surface being internalized in the erythrocyte [10]. We examined whether a sphingomyelinase.induced endovesiculation in NPC fibroblasts could account for the observable decrease in the size of the cholesterol oxidase susceptible pool of cholesterol in sphingnmyelinase-treated cells. in contrast to the findings in erythrocytes, we were not able to demonstrate increased endovesiculation, at least when determined from the rate of fluid-phase pinocytosis of [3H]sucrose. Although not statistically significant, it appeared that sphingomyelinase treatment of NPC fibroblasts actually resulted in a slightly decreased rate of [3H]sucrose uptake. Thus it appears unlikely that endovesiculation of the plasma membrane was the operating mechanism that was responsible for the flux of cholesterol from the cell surface into intracellular compartm~ts in rcspon~ to sphingomyelin degradation. Where is the lesion in Niemann-Pick type C fibroblasts? The mutant cells display a clear defect in the acute r~ponses to LDL uptake, but when intracellular cholesterol transport is forced by sphingomyelinase treatment these cells respond like normal fibroblasts in the various metabolic responses studied so far. Recent studies by Tabus and co-workers [14 have suggested that, at least in the macrophage model, the cholesterol in the plasma membrane pool is a preferred substrate for the ACAT-catalyzed esterification reaction, it is then possible that LDL-derived cholesterol, mostly liberated by hydrolysis of cholesteryl esters in lysoseines, is transported first to the cell surface and only later is diverted into the endoplasmic reficulum, where it becomes esterified by ACAT. In Niemann-Pick type C, it is possible that transport of cholesterol from lysosomes to the plasma membrane is normal, but that the the further spontaneous transport from the plasma membrane to the intracellular

regulatory pool of cholesterol is sluggish. With a continuo,~ls uptake of lipoprotein-derived cholesterol from the ext;acellular space (both receptor-mediated and -independent uptake), it can be envisioned that a sluggish release of cholesterol from the plasma membranes into NPC cells would lead to an initial accumulation of unesterified cholesterol in the plasma membranes and eventually also in lysosomes. This speculative sequence would be compatible with findings showing that NPC fibroblasts, when exposed to LDL accumulate unesterifled cholesterol, some of which can be found as filipinstainable sterols in lysosome-like organelles [17]. Since movement of cholesterol from the plasma membrane to intracellular membranes can be effected with sphingomyelin depletion, as this study shows, it is not likely that a putative intracellular carrier of cholesterol is missing in Niemann-Pick type C fibroblasts. However, it is possible that the plasma membranes of NPC fibroblasts in the native state have some properties that lead to a slower release of cholesterol from the cell surface into the cell. If this were true, then native NPC fibroblasts should also display a decreased efflux of cholesterol from their plasma membranes to extracellular accepters (non-receptor mediated). Indeed, we now have preliminary data indicating that efflux of radiolabelled cholesterol from plasma membranes to HDL 3 is decreased in NPC fibroblasts compared with normal fibroblasts [18]. Acknowledgements The skilled assistance of Karin Sundquist and Maria Culala in early parts of this study is warmly acknowledged. This study was supported in part by the American Heart Association (WA Affiliate) and by NIH Grant HL 18645. Dr Slotte received supporting grants from Lit~lke-Farmos (Turku), The Borg Foundation (Abe Akademi), the Ella and George Ehrnrooth Foundation (Helsinki), the Oscar Oflund Foundation (Helsinki) and the Foundation of the Turku City Council. References ! Brady, R.O. (1983) in The Metabolic Basis of Inherited Disease (Standbury, J.B., Wyngaarden, J.B., Fredrickson, D.S., Goldstein, J.L. and Brown, M.S., eds.), pp. 831-841, McGraw-Hill, New York. 2 Pentchev, P.G., Comly, M.E., Kruth, H.S., Tokoro, T., Butler, J., Sokol, J., Filling-Katz, M., Quqirk, J.M., Marshall, D.C., Patel, S., Vanier, M.T., and Brady, R.O. (1987) FASEB I, 40-45. 3 Pentchev, P.G., Comly, M.E., Kruth, H.S., Vanier, M.T., Wenger, D.A., Patel, S. and Brady, R.O. (1985) Proc. Natl. Acad. Sci. USA 82, 8247-8251. 4 Pentchev, P.G., Kruth, H.S., Comly, M.E., Butler, J.D., Vanier, M.T., Wenger, D.A. and Patel, S. (1986) J. Biol. Chem. 261, 8775-16780. 5 Slo~to, J.P. ~.-,d Bierman, E.L. (1988) Biochem. J. 250, 653-658.

309 6 Slotte, J.P., HedstriSm, G., RannstrSm, S. and Ekman, S. (1989) Biochim. Biophys. Acta, in press. 7 Slotte, J.P., Chait, A. and Bierman, E.L. (1988) Arteriosclerosis 8, 750-758. 8 Heider, J.G. and Boyett, R.L. (1978) J. Lipid Res. 19, 514-528. 9 Low~, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 10 Allan, D. and Walklin, C.M. (1988) Biochim. Biophys. Acta 938, 403-410. 11 Liscum, L. and Faust, J.R. (1987)J. Biol. Chem. 262, 17002-17008. 12 Lang¢, Y. and Ramos, B.V. (1983)J. Biol. Chem 258, 15130-15134. 13 Slotte, J.P., Oram, J.F. and Bierman, E.L. (1987) J. Biol. Chem. 262, 12904-12907.

14 Tabas, I., Rosloff, W.J. and Boykow, G.C. (1988) J. Biol. Chem. 263, 1266-1272. 15 Patzer, E.J., Wagner, R.R. and Barenholz, Y. (1978) Nature 274, 394-395. 16 Moore, N.F., Patzer, E.J., Barenholz, Y. and Wagner, R.R. (1977) Biochemistry 16, 4708-4715. 17 Sokol, J., Blanchette-Mackie, E.J., Kruth, H.S.~ Dwyer, N.K., Amende, L.M., Butler, J.D., Robinson, E., Patel, S., Brady, R.O., Comly, M., Vanier, M.T. and Pentchev, P.G. (1988) J. Biol. Chem. 263, 3411-3417. 18 Skogster, S. and Slotte, J.P. (1989) 30th International Conference on the Biochemistry of Lipids (Abstract).