Biochhnica et Bivphysk'a Acre, t 126(1992) 291-297

29 I

~ 1992Elsevier Science Publishers B,V. Alf rig.hts reserved 0OI)5-27(dl/92/$05.tX)

BBALIP 53946

Modulation of sphingomyelinase-induced cholesterol esterification in fibroblasts, CaCo 2 cells, macrophages and smooth muscle cells O.

Stein ", M. Bcn-Naim ", Y. Dabach ~', G. Hollander b and Y. Stein

t,

"Depanmem of h~ff~,rimental Medichle and Cancer Reseurch, th,brew Unh't'rdt.v.Hadassah Medical Sch~nd and t, Lipid Research Lalu~ratoB.; Dcptlrtment of Medichw, Hadassah Unil'ersity Hospital, Jerusalem (Israd)

(Received I1 March It~92)

Key words: Sphingomyclin;Sphingomyclinase:ACAT; Brefeldin: Verapamil; ChLflcstcroltransport; (Human skin fibrobLast); (CaCo~ cell); (Mouse macrophage): (Aortic smooth musclecell) The present study has fi~cused on three questions concerning the effect of sphingomyelinase on release of free cholesterol from the plasma membrane and its inlracellular translocation: (i) Can one change the direction of the flow of cholesterol? (it) Can one modulate the flow? (iii) May such a mechanism be relevant in a~herogenesis? (i) The results obtained show that even in the presence of potent mmlipoprotein cholesterol acceptors in the medium, the intracellular flow of cholesterol is not reduced as measured by cholesterol esterification. Moreover, in sphingomyelinase-treated cells, cholesterol efflux in presence uf nonlipoprotein acceptors was not enhanced even when intracellular ester(flea(ion was inhibited. (it) Modulation of the sphingomyelinase induced cholesterol flow can be obtained by I(~] pM verapamil which reduces il. In human skin fibroblast, interference with the delivery of free cholesterol to its site of ester(heat(an was fimnd in the presence of brefeldin A. (iii) Aortic smt~th muscle cells in culture are sensitive to low concentrations of sphingomyclinase and the increase in ester(fled cholesterol is evident also alter cxpt~sure to the enzyme for 24 h. The present results suggest that in the plasma membrane, free cholesterol bound to sphingomvclin may be in a compartment which renders it more available for transport to the cell interior Lhcn for cfflux, in view of the sensitivity of aortic sm¢~)th muscle cells to sphingomyellnase, this mechanism fi~r enhanced csterihcation of cholesterol could be relevant to the transfi)rmation of arterial smooth muscle cells into fi~am cells in the process of athcrogenesis.

Introduction In most cells, the bulk of cellular free cholesterol is located in the plasma membrane where it is intercalated between phospholipid molecules of the bilayer, it is thought that its main function is to stabilize the cell layer and to prevent the leakiness of the cell. Among the plasma membrane phospholipids, sphingomyelin has the highest affinity for free cholesterol [|,2]. Previously we have studied in tissue cutture systems the effect of phospholipid liposomes added to the medium on the cfflux of free cholesterol from cells [3-5]. Sphingomyelin liposomes were found to deplete free cholesterol from different cells, especially when complexed with delipidated H D L apoprot¢ins. Re-

Correspondence: Y. Stein, Lipid Research Laboratory, Department of Medicine, Hadassah University Hospital, P.O. Box 1222(I. Jerusalem 91120, Israel.

cently, Slotte et al. have shown [6-8] that addition of a neutral sphingomyelinase to human 6broblasts and other cell types causes hydrolysis of plasma membrane sphingomyelin and translocation of free cholesterol into the ceil interior which is followed by its esterification. The aim of the present study was to [earn more about the transport of the free cholesterol after sphingomyelinase treatment; whether its flow may be channelled to the exterior and how it will be affected by agents known to interfere with intracellular traffic, Drugs were chosen, such as verapamil which impairs the translocation of free cholesterol from lysosomes to endoplasmic rcticulum [9], and brefcldin A, known to disrupt vesicular transport from endoplasmic reticulum to the Golgi apparatus without impairment of the flow of free choles'~,tol from endoplasmic rcticulum to plasma membrane [10]. In addition, we investigated also the susceptibility of different cells to the induction of cholesterol ester(heat(on by sphingomyelinasc, in particular those that participate in cholesterol absorption or are involved in atheroma formation.

292 Materials and Methods

Cell culture Human skin fibroblasts (HSF) from healthy donors and CaCo, cells were grown in Eagle's MEM medium supplemented with 10% fetal bovine serum. Bovine aortic smooth muscle ceils were prepared as described before [11] and were cultured in Dulbecco-Vogt medium supplemented with 10% fetal bovine serum. All cells were seeded in 35-mm petri dishes and cultured for 8 days with three medium changes. In order to label the cells with [3H]cholesterol, the latter was added to serum containing medium (1 ~tCi/ml) and the cells were grown in the labeled medium from the time of seeding to obtain complete equilibration of the different cellular cholesterol pools. Periumeal macrophages were obtained from mice 4 days after intraperitoneal injection of thiogly~.ollate and were cultured for 24 h in 35-mm petri dishes with MEM containing 10% fetal bovine serum and [3H]cholesterol [12],

Experimental design 24 h prior to the start of the experiment, the labeled medium was removed, the cell layer washed with phosphate-buffered saline (PBS) and the cells incubated for 24 h in Dulbecco-Vogt culture medium conlainin~ 0.5% serum. Thcieatter, the ceils were washed with Ham F,, medium containing !% bovine serum albumin and serum free Ham Fi¢, medium for 10 rain each at 37°C. Incubations with sphingomyelinase were carried out in serum-free Ham F m medium. Two types of experiments were performed: either continuous exposure to sphingomyelinase for up to 24 h, or short-term exposure (45-60 rain) followed by a wash and post-incubation. The wash was either PBS alone or PBS containing 5 mM EDTA followed by PBS. Post-incubation was carried out in serum free medium with appropriate additions. At the end of the experiment, the medium was collected and the cell layer was scraped with 1 ml 50% methanol and 2 ml 1011% methanol using a Teflon policeman. After addition of an equal volume of chloroform, the lipids were extracted and the deiipidated residue used for determinatic,~, of protein.

Chemical and ctzromatographie procedures Liposomes were prepared from dilinoleyl or dioleyl phosphatidylcholine by sonication at a conce~ltration of I mg/ml [4]. High density lipoproteins (HDL) were isolated Ly ultracentrifugation from human plasma at d 1.063-1.21 g/ml for 48 h [13]. The HDL fraction was delipidated according to Scaau anti L~a~:lstein[14] and is designated apo-HDL. To prepare mixtures of liposomes and apo-HDL the two components were incubated overnight at room temperature prior to addition to cells. Lipids were extracted and purified according to Folch et al. [15]. Separation of 3H free and esterified

cholesterol was carried out by thin-layer chromatography on silica gel p!at.es using the solvent system of chloroform/ethyl acetate (95:5, v/v); for the separation of lipids labeled with [3H]oleic acid, the solvent system used was petroleum ether/ethyl ether/acetic acid (80:20:1 v/v). The labeled compounds were identifle,~ with "~e aid of pure standards as visualized by iodine vapors, the areas were cut and counted. Radioactivity was determined with a Tricarb//-scintillation spectrometer (Minoxy, Packard) with absolute activity analyser. Protein was determined according to Lowry et al. !16] and lipid phosphorus according to Bartlett [17].

Materials [9,10{n)JH]Oleic acid, [7(n)JH]cholesterol were from Amersham International (UK). All culture media and bov;;e fetal serum were from Gibco (Grand Island, NY, USA). Brefeldin A was obtained from Epicentre Technologies, Madison, WI, USA. ACAT inhibitor, compound 58-035 (3-[decyl-dimethylsilyl]-N-[2(4-methyl-phenyl)-l-phenyl-ethyl]prop~,namide) was generously provided by qandoz Incorporated (East Hanover, NJ, lISA). Verapamil was obtained from lkaPharm, Jerusalem, Israel. Sphingomyelinase from Staphylococcus attreus and bovine serum albumin were obtained from Sigma Chemicals, St. Louis, MO, USA. Results

In the first experiments, we enquired whether efflux of free cholesterol from cells will affect the sphingomyelinase-induced intracellular cholesterol esterification. Tilerefore, the cells were exposed to low and high concentrations of sphingomyelinase in the presence of mixtures of delipidated HDL and phosphatidyl choline liposomes. The data presented in Table ! are from a representative experiment repeated several times and show that, notwithstanding the loss of up to 7% of the cellular ['~H]cholesterol, the accretion of ['~H]cholesteryl ester induced by sphingomyelinase reached the same 7-fold level as in sphingomyelinasetreated cells not exposed to cholesterol acceptors (Table I). This experimental design, which precluded cholesterol exchange, permitted also the investigation of whether free cholesterol efflux does increase in sphingomyelinase treated cells. Although ['~H]eholesterol efflux from HSF increased markedly in the presence of phosphatidylcholine liposomes and more so with mixtures of apoHDL and phosphatidylcholine liposomes, no additional cholesterol efflux was obtained in the presence of low or high concentrations of sphingomyelinase (Table i). The next question was will free cholesterol efflux, in presence of acceptors in the medium, increase in sphingomyelinase-treated cells in which chc~lestero[ esterification had been inhibited.

293 TABLE !

TABLE I11

Sphlngomyelinase-ituhtced ('t)ok,,~terot esterificatkm and fi'ee choh,,~terol efflm ha human .tkhl fihrahla.ws (HSF) bt the prt:vence of chub'stere/ atTt'ploL~" bl medinm

F4[ect of i,¢rapltttdl ell slJhingomyelinu,~('.imha'ed e.vterijh'ati, n ~)1" ] "~Hh'hok'stend hi ItSF

Conditions: Human IISF were labeled with [314]cholesterol from the lime of seeding. After 6 days, the cells were washed twice with PBS and incubated flu 24 h in medium contain{n8 (1.5'~ serum. On the day of the experiment, the cells were washed )5 min with Ig: albumin in F,) medium and 15 rain in F,) without serum ill 37~C. The cells were c~)x)sed fi)r f) h to sphingomyclinase alone or togelhcr with 160 /,tg/ml of delipidatcd HDL (Ape HDL) or 80 ~ g / m l of phosphatidylcholine (PC) llposomes, or a mixture of bath 160 ~g:8() /zg (ApoHDL/PCL At the end of incubation, the medium and cells were eoilecled and free and eslerified cholesterol (FC and CE) were determined as described in Materials and Me{buds. Tot:d cellular radioactivity was 1.5,11)5 dpm/dish. Values are means±S.E, of triplicate dishes. Sphingo- Additions

¢~ of cellular label

myelinase (mU/ml)

[~HICE ia celts I~I~]FC ~.f~lu^ none Apol ! DL PC liposome~ AI'r)HDL: PC liposomes

I. I ± 0.01 1.2 ± 0.0) 1.3 +_0,01 1.4 ± (L2

0.3 + 0.01 0.9 + 0.02 4.3 _+0.5 6.9 ± 0.2

none Apol I DL PC liposomcs ApoltDL: PC liposomes

7>J ± 0.9 7.5 ± 0.2 7.8 ± 0.7 7.0_+0.1

0.4 + O.(ll 1.5 ± 0.3 4.3 ± I). I 6.4 ± I).l

none ApoHDL PC fiposomes ApoHDL: PC lipost)mes

6,1 ±0.2 6.1 + 1).7 7.6 ± I).2 7.0 + 0.2

t).4 +_I).l 1.3 ± (L 1 3.{l_+0. I 7.2 ± 0.2

12.5

fiX)

The sphingomyelinase-induced cellular cholesteryl ester increase was prevented almost completely (81-98%) by the addition of an ACAT inhibitor (Sandoz compound 58-035) [6], yet the [~H]cholesterol efflux in presence of phosphatidyleholine liposomes remained unchanged (Table !I).

eruditions: as in Table I. Total cellular cholesterol was 2" 11)~-3 • 105/dish: the¢; of ['~lt]chnlesleWI ester ([3H~'E)in cells not Ireated with sphingomyelinase ~as 1.8+_0.14, 1.2_+l).15 and 2.0+t).l i~ Expt 1, 2 and 3, respeclively. Values arc means ± S,E. of triplicate dishes in each experiment, a vs. b and c vs. d, P < 0.01, Sphingu-

Vcn.pamil

f/t of cellular lal~l

myelinase (mU/ml)

(#M)

t~lt]CEincetls

[~lt]FCefflux

8.5 + 0 . 4 8 f ) , l).3

0.44 ± 0 , 0 5

Expt,

I0

tl 50

2

It) I(I

0 I()0

9.3±0.4" 5.8 :[: O.t) ~'

[l.70 ± [).01 037 4:(),t)2

3

511 50 50

l) 50 lift)

4,t +~(I.3 ~ 3,1 + (1.3 23 _+0.1 d

1L72 ± 1l,(15 l).Tll_+_0.08 0.80± t).{)5

i .....

I0

0,38 + (}.01

In a previous study w e have shown that addition of verapamil (a Ca -~* channel blocker) to cultured cells resulted in decreased cholesterol ester{flea{ion; thus, it was of interest to learn whether the drug will interfere with sphingomyelinase induced cholesterol ester{flealion. Table Ill shows that at high concentrations of verapamil (10() p:M), sphingomyelinase-induced cholesterol ester{float{on was reduced only by 34-38%, lower verapamil concentrations (50 ~M) were marginally or not inhibitory. Verapamil had no effect on [3H]cholesterol efflux in absence of cholesterol accepters. Another parameter investigated was whether one can enhance the sphingomyclinase-induced cholesterol ¢sterification. In CaCo, cells, addition of brefeldin A (BFA) caused an increase in cholesteryl estcr apparently by activation of the ACAT reaction [18]. However, in sphingomyelinase-treated HSF, addition of BFA resulted in a reduction of cholesterol esterifica-

TABLE I1

Sphingamyclinuse.imhwcd chotestcnd e.~'teri]icaticm and [ree cholesterol t¢fl, r in ItSF in pn'.wnce of ACA 7' inhihitor and pho.v)hatklyk'holOte (P('i

liposot)lCS Conditions: as in Table I. The concentration of sphingomyeliuase was 25 mU/mt and Ill mU/ml in Expl. I and Expt. 2, respectively: the concentralion of ACAT inhibir)r was 1.6/~g/ml and of PC liposomes 75 # g / m l and incubation was 6 h, Total cellular radioactivity was 2,5- iI) ~ dpm/dish. Values are means + S.E. of triplicate dishes in each experiment. Sl)hingomyelinase

ACAT inhibtier

PC liposomcs

'~ (If cellular label Expl. l

Expt. 2

[)tt]CE in cells

I~II]FC efflux

[ Lll]CE in cells

I3]I]FC efflux

+

-

-

6.3 _+ 0.5

0.4 ± 0,05

4.8 ± (4.2

I).5 +_ (k()l

+ + +

+ +

+ +

7. t ~ (I.7 1.9 ± I). I 2.8 :l:il,4

4.3 ± t).3 0.4 .+_0.03 4,2:+: 0,1

-

-

-

1.8 + 0.3

0,3 +_ (l.(15

5.(14=_11.2 1.3 ± O. I 1.9_-t:O.I 0.5 :[- 0.01

3,4 +_0.2 0,5 ± O.(IS 3.6±0.t 0.5 ~ ().(15

294 T A B L E IV

TABLE V

Effect of brefeldin A (BFA) on .~phhlgomyelinase induced eslerij~cation of ['*H]choh,sterol in HSF

Effl,ct of lime af additioa of brefehlin A on sphhtgomyelinase.iadlwed /"it/cholesterol esreri[ication br HSF

Conditions: as in Table !. The cells were incubated for 15 h with 511 m U / m l of sphingomyelinase and BFA, 0.66 9 g / m l . in Expt. ! and 1 p g / m l in Expt. 2, Total radioactivity was 2,5" !0 s dpm/dish. Values are means +- S.E. of triplicate dishes in each experiment, a vs, b and e vs, d. P < 0,01.

Conditions: Cells were cultured and labeled as in Table I. The cells were exposed to sphingomyelinase (50 m U / m l ) for 611 rain and then the enzyme containing medium was removed and the cell layer washed and incubated for additional 5 h. BFA, 1 ~ g / m l was added either I h prior to ( = - ! h) or together with sphingomyelinase ( = 0 h) or after removal of the enzyme. Data are % increase of l~H]cholesteryl ester (['~H]CE) as compared to cells not treated with sphingomyclinase in which there were 2.2.1115 dpm/dish of t3Hkholesterol, of which 0.t17 +_0,07% and 2.0~0.2% were in cholesterol ester in Expt. 1 and Expt. 2, respectively, Values are means_+ S.E. of triplicate dishes in each e~periment.

Expt.

Additions

['~IttCE (% of cetlular label) with sphingomyelinase

without sphingomyetinase

I

None BFA

4.0 +- 11.3 " 2.5 +- 0, I ~`

0.95 +- 0.02 0.96 +- 0,01

2

None BFA

4.1

+0,2 " 2.0 _+O. 1 a

0.81 +0.0I 0.90 ± 0.01

tion by 38-50%, while in ceils not treated with sphingomyelinase, there was no effect of BFA (Table IV). To determine the time frame of the inhibitory effect of BFA on cholesterol esterification, the following experiment was performed. BFA was added either prior to or together with sphingomyelinase or ~tt various time intervals after removal of the enzyme, and the cells were incubated for 5 h after enzyme removal. The marked inhibitory effect of BFA on sphingomyelinase-induced [~ H]cholesteryl ester was seen when the drug was added either I h prior to or together with the enzyme or even 1 h after enzyme removal (Table V). Since BFA inhibited the sphingomyelinase-induced cholesterol esterification in HSF, we repeated the experiments in CaCo, cells which have a more active eholesteryl ester turnover, in Expts. 1-3, the cells had been labeled with [~H]cholesterol since seeding, in Expt. 4, [~H]oleic acid was added at the beginning of the 6 h incubation with sphingomyelinase (Table Vi). In all experiments, sphingomyelinase treatment resulted in a 2-3-fold increase in the recovery of labeled cholesteryi ester in the CaCo: cells. In analogy to the

TABLE

Sphingomyelinase

BFA added in relation to sphingomyelinase (h)

[~aIt]CE in cells (% of nonlreated cells) Expt. I Expt. 2

+ + + + +

- i 0 + i +3 -

100 223+ 10 226+_ 8 300+- 8 357 4-_ 7

100 170+-20 160 + 311 250+10 270 _ 211

findings with HSF, |00 /~M vcrapamil reduced the sphingomyelinase-induced increase in ['aH]cholesteryl ester from 224% of control value to 122% (Expt. 1) and 272% of control to 142% (Expt. 2). In contradistinction to the effect in HSF, addition of BFA to CaCo 2 cells resulted in enhancement of the sphingomyelinase-induction of ['~Hkholesteryl ester accre tion. Thus, in the ['~H]cholesteroi-labeled ceils, [~H]cholesteryl ester increased 6.2-7.5-fold over the control value; and 3.7-fold in the [3H]oleie acid experiment. Addition of BFA without sphingomyelinase caused a 3-4.5-fold increase in [~H]cholesteryl ester (Table VI). Another question investigated was whether the sphingomyelinase-induced [~H]cholesteryl ester is metabolized rapidly in CaCo, cells, as was described

VI

E[fect of rc,rapamil and br~f(,Idin A {BFA~ on sphing4mlvelit a.w.imhwed t,,sterification of/~lt]chol,'.~fero! i:t CaCo : celh' Conditions: CaCo: cells were labeled with [~Hkh(flcslerol and radioactivity was 6.10"~-1 • 10" dpm/dish. In Expl. 4. nonlabeled radioactivily was 1.3' 10" dpm/dish. The concentration of BFA concentralion of verapamil was i00 p M. Values arc ntcans + S,E. Sphin'gomyelinase (mU/ml)

Verapamil

BFA Expl.'i.:

further treated as HSF described in Table 1 (Expts. 1-3). Total cellular cells were incubated fl)r 6 h with 100 # M [3tl]~deic acid and total cellular was I p,g/ml in Expts, I and 2 and 0.25 ~.g/ml in Expts. 3 and 4, The of triplicate dishes in each experimen|.

I'~HICE in cells (% t)f'cellular'label) I 2

'"

3

4

0

-

-

5.0 ± 0. I

50

-

-

11.2 + 0.4

3.3 __ 0.2 9.5 __+0.6

3.4 + 0. I I0.8 +-0.4

2.8 +__0.2 5,5 + 11.3

50 50 0

+ -

+ +

f~.~ :J:.0.1 31.8 + (1.2 I 7.7 +- t1,4

4.7+0.1 24.6 ~ ().9 -

25.6 + (1.2 15.5 4-__0.2

10,3 4. 0.2 8,6 4. O. I

295 TABLF VII Fate of sphingon~yelinase-induced ['¢HICE in CoCo 2 cells daring pro. longed chase

Conditions: CoCo: cells were labeled with 13Hkhulesterol and treated as HSF, describedin Table I. The cells were then exposedto sphingorayelinase,50 raU/ml for 45 rain. Thereafter, the enzyme was removed, the cell layer washed three times with PBS and the cells were post.incubated in serum-freemediumfor the times indicated. ['~HIChalestewtester ([~HICE)in cells not exposed to sphingomyelinasewas 2.7±0.1 and 5.8±0.2~ of cellular label in Expt. I and 2, respectively.Cellular label was 3" IOs dpm/dish. Values are means±S,E. of triplicatedishes in each experiment. Sphingomyeliaase

Postincubation

['~H]CEin ceils (~. of nontreatedcells)

(45 rain)

(hi

I

2

-

-

100

!00

+ + + + + + +

o 1 2 3 6 8 18

174_+ 3 285± 4 370± 15 363± 7 400 ± I 1

! 18:t:8 278:t:8 284+_4 262+ I I 256+ 10 1164-6

-

-

for BHK cells [7]. Results presented ia Table VII show that in pulse chase experiments, [3H]cholesteryl ester increased up to 2 h after sphingomyelinase removal and the high values persisted for ~ h. Since accretion of cellular cholesteryl ester is one of the hallmarks of atheroma, we investigated the effect of sphingomyelinase in aortic smooth muscle cells and in macrophages. Results of representative experiments are seen in Table VIii, and tt appears that in TABLE Viii Comparative effi,cl of sphilagomyelinase on chotesk'rol esterification in lmrine aortic smooth mu.~de cells (smooth muscle cells) and mouse macrophages

Conditions: I~vine aortic smooth muscle cells were labeled and treated as HSF de~ribed in Table I. Mouse peritonealmacrophages were labeled with [~H]cholesteral for 24 h, incubated in medium containing 03~ serum for 3II rain. washed with 1% albumin and serum-free mediumas in Table I. Valuesare means±S.E, of triplicate dishes in each experiment, a vs. b, P < 0.002: ¢ vs. d, P < 0.01. Sphingomyelinase (mU/ml) 0 12,5

25 o 50,o

Hours

['~H]CE (f~ of cellular label) SMC raacrophages

6 24

1.2_0.03" 1.7 + 0.05

1.2+0.I ~" 1.8 + O, I

3.5 + 0.05 D,

2.0-I-0.I d

6

24

6.2 ± 0.2

1,8 ~:0,03

6

3.3 + 0.2

2. I + 0.07

24

6,2±0.2

1,8±0,I

6 24

3.3 + 0.05 6.1 ±0,I

3.! :i:O. I |.7±0.7

macrophages, 6 h treatment with up to 25 m U / m l of sphingomyeiinase resulted in a 70% increase in cellular ['~H]cholesterjl ester; with 50 m U / m l , the increase was 160%. However, when the cells were exposed to sphingomyelinase for 24 h, no increase in ['~H]cholesteryi ester over the control values was found. A different pattern of response was encountered in smooth muscle cells. Maximal increase in [-~Hkholesteryl ester pccuffed already with the lowest enzyme concentration used; it was 3-fold after 6 h and 3.5-fold after 24 h, Discussion

In the present study we attempted to evaluate whether marked efflux of free cholesterol, obtained by addition of mixtures of phosphatidylcholine liposomes and delipidated HDL, will affect the sphingomyelinase-induced cholesteryi ester accretion. The results have shown unequivocally that even when a considerable efflux of free cholesterol was achieved no decrease of cholesterol esterification occurred. There are several possibilities to explain this phenomenon: (it that the sphingomyelinase released an excess of free cholesterol from the plasma membrane; (it) that the action of the sphingomyelinase is much more rapid than the efflux of free cholesterol from the plasma membrane to the accepter in the medium; (iii) that the affinity of the intracellular transporting system for the less tightly bound cholesterol in plasma membrane (after sphingomyelinase hydrolysis) is much higher than that of the cholesterol accepter in the medium; (iv) that there are two functional pools of free cholesterol in the plasma membrane, one intercalated between phosphatidylcholine molecules and released to the exterior when accepters are present and one which is available for the intraceilular transport system, when sphingomyelin is cleaved into ceramide and phospho. choline. The latter possibility is supported perhaps by the finding that in mixed monolayers of cholesterol and sphingomyelin, cholesterol condenses sphingomyelin more than phosphatidylcholine [19] and that the resistance to oxidation of cholesterol by cholesterol oxidase is increased in sphingomyelin monolayers [20]. Additional support for the hypothesis that there are two functional pools of free chole>aerol within the plasma membrane can be derived from the experiment with the ACAT inhibitor, in which sphingomyelinase treatment did not increase the efflux of free cholesterol into the medium in the presence of cholesterol accepters. These results are in agreement with those of Slott¢ ct al. [8] who added HDL 3 to [ 3H]cholesteroblabeled skin fibroblasts and found no effect of sphingomyelinase treatment on the loss of radioactivity into the medb~m. Another study was addressed primarily to the elucidation of the mechanism by which sphingomyelinase treatment inhibits HMG-CoA reductase activity [21],

296 Pretreatment of r;a inte.,,.'ina[ epithelial cells with din. lcylphosphatidylcaoline vesicles or HDL.~ not only abolished the inl'ibitory effect of sphingomyelinase on HMG-CoA redu~'tase activity, but s!,.'me[a',ed the c.nzyme activity rci,,ardless of the presence of sphingomyclinase [211. It should be noted, however, that the full reversal of the inhibitory effect of sphingomyelinase on HMG-CoA rcductasc activity was evident only with 500 .aM phosphatidylcholine vesicles, but was only marginal with 250 .a M. This concentration of phosphatidylcholine (500 .aM) prevented also sphingomyelinase-induced cholesterol esterifieation [21], in contrast to our experiments in which only 100 .aM phosphatidyleholine was used. In human skin fibroblasts and CaCo 2 cells, tae sphingomyclinase induced c[a:lcstcryi ester accretion was moderately reduced by high concentration of verapamil 1100 ~M). In experiments in macrophages in which cholesteryl ester synthesis was stimulated by addition of acetylated LDL or fl-VLDL, it was reduced markedly by 25-50 p.M verapamil and this was not due to a direct effect of verapamil on ACAT [9]. This effect of verapamil was also seen in bovine or rabbit aortic smooth muscle cells in which cholcstcryl ester synthesis was induced by addition of LDL to the medium [9]. The lesser effect of verapamii in the present cxpcriments would be in line with the hypothesis we proposcd in the previous study [9], namely that the cholcstcryi ester is hydrolyzed in the iysosomal compartment but the free cholesterol remains sequestered there and, theretbre, does not reach the ACAT located in the endoplasmic reticuIum. In the present study, the frec cholcsterol released from the plasma membrane bypasses the [ysosomes and reaches the ACAT compartment directly. One might envisage the possibility that verapamil could reach higher concentrations in iysosomes than in the plasma membrane and therefore have a ie~s pronounced effect under the pre,~ent experimental conditions. Brefeldin A is a lipophilic fungal product which has a high affinity for some proteins found in the Golgi apparatus. The curliest interaction between BFA and Golgi proteins occurs after 31) s and results in a release of a l l0 kDa protein [22]. This protein (¢ICOP) was identified as one of four subunits that form the nonclathrin coat in the absence of which cessation of the forward flow of membranes from endoplasmic reticulure to the Golgi may occur [2~]. In a previous study, we have shown in CaCo, cells that cholesteryl ester accretion occurs after addition of BFA and that this was not due to direct activation ,~f ACAT by BFA [18]. These findings were corroborated also in the present study. It appears that ACAT may not have been saturated with substrate after BFA treatment, since when sphingomyclinase induced an additional flow of free cholesterol from the plasma membrane to the endo-

plasmic reticulum, cholesterol cstcrification was further enhanced. In human skin fibroblasts, BFA interfeted partially with the sphingomyclinase induced ~:hoicsterol esterification. One possible expianation could be that in this cell following sphingomyelinase treatment, some of the cholesterol is delivered to the cell interior by a vesicular transport [24] sensitive to BFA. In a recent comprehensive review [24] on lipid transport in eukaryotic cells, il was concluded that "a single unifying model for intracellular transport of lipids cannot account for all the existing data on lipid movement within cells. Instead, multiple pathways of intracellular lipid transport have been suggested depending on molecular species, cell type and membrancs under consideration". Thus, in the plcscnt study, BFA reduced the flow of free cholesterol from the plasma membrane into the cell interior in sphingomyelinasetreated HSF, while cholesterol flow from endoplasmic reticulum to the plasma membrane was not affected by BFA in Chinese hamster ovary cells [10]. Cholesterol esterification in bovine aortic smooth muscle cells induced by sphingomyelinasc continues for up to 24 h and could bc of some interest in unravelling the riddle how smooth muscle cells transform wlthin the aortic wall into foam cells. The explanation for this well-described pathological finding eluded us till now, nothwithstanding years of research by many investigators. It is known that some cholestcryl ester formation in smooth muscle cells can be induced by direct transfer of free cholesterol from free cholesterol-enriched LDL [25]. In the athcroma, disintegrating macrophages could release a sphingomyclinase that in turn could act on thc plasma membrane of smooth muscle cells and cause cholestcryl ester accumulation. Experiments are now in progress to determine whether such a mechanism will lead to the development of smooth muscle cell-derived foam cells in the tissue culture system.

Acknowledgements This study was supported in part by the Mario Shapiro Fund, The Hebrew University, Jerusalem. The excellent secretarial help of Mrs. J. Hollander is gratefully acknowledged.

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