Biochimica et Biophysica Acta. 1085( 1991) 1-6

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© 1991 ElsevierScience Publishers B.V. (h')05-2760/tJI/$03.50 ADONIS 000527609100219X

The effect of chok;sterol on the accumulation of intracellular calcium Q i Z h o u , S h i r o Jimi, T e r r a n c e L. S m i t h a n d F r e d A. K u m m e r o w BarnsMes Research Laborato~. . Department of Ftu~d Sctenct'. Unirersity of Illinois. Urbana. IL (U.S.A.)

(Received 26 November19~)) (Revised manJscript received 28 February 1991)

Key words: Cholesterol/phosphatidylcholinelip~Jsome;Smoothmusclecell; Intracellularcalcium;(Ca2÷+ Mg2~)-ATPaseactivity Cholesterol/egg phosphatidylcholine (PC) liposomes (1 : 1 or 4: I, M / M ) , in which the absolute amount of PC was adjusted to be the same, were incubated with cultured bovine arterial smooth muscle cells for up to 8 h at 3TC. The effect of increased cellular cholesterol on the accumulation z~' intraceilular calcium in these cells was studied. The results indicate that the intracellular calcium content, measured by F u r a - 2 / A M , was increased 2.3-fold by incubation with 4:1, cholesterol/PC liposomes. Kinetic analysis using 4SCa2"~ indicated that the increased calcium influx was due to increase of pool size, not from a change of rate constant. (CaZ++ MgZ+)-ATPase activity was decreased by 4:1, cholesterol/PC liposomes. The molar ratio of cholesterol/phospholipids in the cell membranes was directly proportional to that in liposomes. No change in phospholipid composition was noted. We suggest that the accumulation of intracellu.~ar calcium was a composite result due to the altering effect of inserted cholesterol on surface area, and to direct interactions between cholesterol and the proteins of the Ca z+ channel and (Caa÷+ Mg 2 ÷)-ATPase.

Introduction The pathological changes involved in atheroselerotic disease include proliferation of smooth muscle cells, lipid deposition and calcification. In the study of these three characteristics much evidence supports the theory that intracellular calcium accumulation is accompanied by a rise of cholesterol in the cell membrane [1,2]. This implies a causal relationship between cholesterol and aortic calcification. Cholesterol, as one t;f the major constituents in biological membranes, can influence membrane properties. The location and functions of cholesterol in natural and artifical membranes have been extensively studied. Although many functions of cholesterol are not well understood, there is general agreement that the major roles of cholesterol in plasma membranes appear to involve minimizing permeability to ions and small molecules [3,4] and modulating physical and me-

Correspondence: F.A. Kummerow,BurnsidesResearch Laboratory, Universityof Illinois, 1208 W. PennsylvaniaAve.. Urbana. IL 61801, U.S.A.

chanical properties of the membrane [5,6]. These effects of cholesterol come from cholesterol altered membrane fluidity [7-9] or from cholesterol modulation of the activity of membrane-bound laroteins [10121. The mechanism by which cholesterol content of the cell membrane correlates with intracellular calcification remains obscure, it is known that cholesterol can increase intraceUular calcium in erythrocytes, probably due to a direct stimulation of t:,e Ca 2÷ channel of the membrane [ 13]. Other data shows that the alteration of Ca 2+ flux by cholesterol is accompanied by a change of membrane fluidity [14]. In a study of (CaZ÷4- MgZ+) ATPase from sareoplasmic reticulum, it was shown that the inhibiting effect of cholesterol on the enzyme was due to the direct interaction of cholesterol on the enzyme [15] and not a change of membrane fluidity induced by cholesterol [16,17]. It is not known whether the inhibition of (Ca2÷ + Mg 2+)-ATPase was accompanied by an increase of intracellular calcium. Moreover, most of these experiments were performed with red blood cells and isolated sarcolemma [13,15]. The influence of cholesterol on the intracellular calcium ion content in arterial smooth muscle cells (SMCs) remains

2 unknown. The present study was designed to investigate the effect of cholesterol on SMC calcium accumulation.

t h e total lipid was collected, kept at 4°C and used on the same day. Just prior to use, 3.3-fold concentrated MEM was added to the suspension adjusting it to the standard ion concentration of MEM (pH 7.2).

Materials and Methods

Incubation of the cells with liposomes Egg phosphatidylcholine (PC) obtained from Avanti Polar Lipid produced a single spot on two-dimensional thin-layer chromatography (TLC) with: (a) chlorof o r m / m e t h a n o l / a m m o n i a (65:25:5, v/v) and (b) chloroform/acetone/methanol/acctic acid/water (6:8: 2 : 2 : 1 , v/v). Cholesterol was obtained from Sigma Chemical. it exhibited a single spot on TLC with hexane/diethyl ether/acetic acid (85:15:2, v/v). All lipids were stored under nitrogen at - 2 0 ° C and were used without further purification. F u r a - 2 / A M (Molecular Probes, OR) was dissolved in chloroform, drizd under nitrogen, and stored at -20°C. Working solutions were prepared immediately before incubation with cells by dissolving stored Fura2 / A M in dimethylsulfoxide (DMSO) and then diluting to 1.5 p.M in a balanced salt solution (BSS) containing 20 mM Hepes buffered to pH 7.4 with Tris base, 135 mM NaCI, 5 mM KCI, 1 mM MgCI 2, 1 mM CaCI 2, 10 mM glucose and 0.025% bovine serum albumin. The final DMSO concentration was 0.15%. 45Ca2+ (Amersham, IL) was diluted to 1 /~Ci/5 /zl with deionized water and stored at 4°C. 45Ca2+ was added to Eagle's Minimum Essential Medium (MEM) (GIBCO, Grand Island, NY) to yield a working solution of 1 tzCi/0.5 ml.

Cell culture Bovine arterial SMCs were prepared and identified by the method of Jimi et al. [18]. The cells were grown to confluence in MEM containing 20% fetal bovine s,~iu,,, (FBS)(3igma) at 37~C in an atmosphere of 95% air and 5% CO: in petri dishes (Coming). When the cells in the dish were virtually confluent, the cells were harvested by using a try~,~in-EDTA (Sigma) solution and plated into 75-cm 2 tissue culture flasks (Coming).

Monolayer cultures of SMCs were grown to confluence in 75-cm 2 tissue culture flasks or in 24-well plates (Corning), and washed three times with MEM without FBS. MEM with or without liposomes was added to the SMCs in the flasks (8 ml) or in the 24-well plates (1 ml), and the cells were incubated up to 8 h at 37°C. The Trypan blue viability of the SMCs was not significantly different after an 8 h incubation with or without liposomes.

Measurement of [Ca 2 +1i with Fura-2 / A M After incubation of the cells with liposomes for periods of 2, 4, 6 and 8 h, the vesicle suspensions were aspirated from the flasks and the cells were washed three times with 8 ml of MEM. Then 8 ml of 1.5/~M F u r a - 2 / A M were added to each flask and the culture was incubated at 37°C for 60 rain. The media was removed from the flasks and the culture washed three times with 8 ml of BSS. The cells were recovered from the flasks by incubating with trypsin (Sigma) for 1 rain and scraping with a rubber policeman. After washing three times with 50 mi cold BSS followed by eentrifugation at 250 × g for 3 rnin to remove trypsin and extracellular F u r a - 2 / A M , the cells were suspended in 7.5 ml of BSS on ice and 2.5 ml of cell suspension were placed in a 1 cm quartz cuvette for fluorescent spectroscopy. The euvette was maintained with continuous stirring at room temperature, and each determination was performed in triplicate. The samples were excited alternately at 340 and 380 nm (bandwidth = 5 nm) light and fluorescent emission intensity was monitored continuously at 510 nm (bandwidth = 5 nm). lntraeellular calcium concentration was calculated from the fluorescence signals as described by Grynkiewicz et al. [20]. Autofluorescence was determined from samples of the cells treated with 15/.d of DMSO without F u r a - 2 / A M .

Liposomal preparation Liposomes were designed to have two cholesterol/PC ratios which were above that found in the native membrane and were prepared by a modification of the method of Inbar and Shinitzky [19]. Solutions of 40 mg of PC mixed with 20 mg cholesterol (1:1, cholesterol/PC, M / M ) , or 80 mg cholesterol (4:1, cholesterol/PC, M / M ) in chloroform were evaporated to dryness under nitrogen and dispersed in 26 ml of deionized water. The dispersions were then subjected to bath ultrasonic irradiation (Bransonie 220, Smithkline) for 40 rain in ice cold water. The sonicated solutions were centrifuged at 21000 × g for 30 min at 4°C. The supernatant which contained about 50% of

Measurement of ~5Ca2+ influx" Confluent ceils in 24-well plates were incubated with liposomes and then gently rinsed three times with MEM at 37*C. 0.5 ml of 45Ca2+ containing medium (1 p.Ci) was added to the wells and the cells were incubated at 37°C for time intervals from 1 min to 240 rain. After removing the radioactive medium the monolayers were washed three times with cold 0.1% phosphatebuffered saline (PBS) containing 1 mM EGTA, and digested in 1 ml of 0.5 M NaOH. An 0.8 ml aliquot of the digested cells was counted in a liquid scintillation counter and 0.2 ml was assayed for protein (Bio-Rad). All determinations were performed in duplicate and

3 tile ~amc experiment was repeateu tr, ree times. The kinetic parameters for the calcium uptake were calculated by the method of Boric [21]. Analysis o f lipids

The basic procedure of Pikul et al. [22] for tissue lipid extraction and measurement was used with modifications. Cells used for the analysis of lipids were washed three times with PBS, harvested by brief trypsin treatment and washed twice with 15 ml of PBS followed by centrifugation at 6 0 0 × g and at 15000×g for 10 min at 4°C, respectively. The fragmented membrane was resuspended in 1 ml of methanol, and sonicated twice for 30 s on ice. Total lipids were extracted by the addition of 30 ml of chloroform/methanol (2 : 1, v / v ) and holding at 4 ° C overnight. Separation of cholesterol m .i phospholipids from the lipid extract was accomplished by TLC on silica gel G plates using petroleum ether/diethyl ether/glacial acetic acid (80 : 20 : 1, v/v). Separation of phospholipids (PL) was by two-dimensional TLC using chloroform/methanol/15 N ammonium hydroxide (65 : 25 : 5, v / v ) and chloroform/acetone/methanol/glacial acetic acid/ water (3:4:1:1:0.5, v/v) according to Nelson et al. [23]. Total cholesterol and phosphorus (Pit in the extract was measured [22], and protein was assayed. A s s a y o f ( C a 2 + + M g 2 +).,4 T P a s e

Preparation of SMC membranes for the assay of (CaZ++ Mg 2+ )-ATPase activity was amended from the procedure of Moore et al. [24]. The SMC monolayers were washed three times with PBS after incubation at 37°C with or without liposomes and harvested by trypsinization. The fragmentation of the membrane was performed by first washing three times with 15 ml of 15 mM Tris buffer (pM 7.4) c-ntaining 1 mM EDTA (neutralized to pH 7.0 v, kh NaP q) followed by centrifugation at 15000 X e for 10 min, and then washing twice with 10 mM Tris buffer (pM 7.4) in the same manner. 1 ml of deionized water was added and duplicate 50 /~1 aliquots of the suspension were used for measurement of protein concentration. The remaining 900 p,I were used to measure (Ca2++ Mg-'+)-ATPase in triplicate following the method described by Moore et al. [24]. Activities were expressed as /.t reel Pi/per mg membrane protein. The data were analysed using a multiple analysis of variance and Seheffe test. Results

Calcium accumulation was significantly greater in the cells pretreated with cholesterol/PC liposomes, as shown in Table 1. The higher the concentration of cholesterol in liposomes, the greater the accumulation of intracellular calcium. The intracellular calcium con-

1AVLm

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Quantitation of the intracelhdar calcium in the SMC~ with or without incubation with Iipostnnes (1 : I and 4:1 cilolesterol: P(_\ M~ M) for 2. 4. 6 and 8 h at 37°C

Values are g~venas mean_+S.E.. Means within the same line with a suDerscriDtletter in commonare statistically different and the letter of a and b repre..ent statistical differences at levelsof P < 0.115and 9.01, respectively lncuba- n Control tion time (hours)

[Ca-""]i (nM) I : 1cholesterol/' 4:1 cholesterol/ PC PC

4 6 s

121.6_+5.5 J 122.2+ 7.9 158.7_+19.7~1 161.1-+24.6 ~,

17 S l(t 13

104.8_+4.7~'b Ii}1S~6.9" 11)8.5±6.2~1~2 105.2+_5.5t'

132.5_+5.0 b 156.5_+17.6~ 162.7~17.0a2 242.7+ 31.1 a.h

tinued to increase with time in these cells. In the cells without prctreatment with liposomes, no notable accumulation of intracellular calcium was found even during an 8 h incubation. In the presence of 1 : 1, cholesterol/PC liposomes, the intracellular calcium concentration increased to ! 16% of control valuc~ after 2 h. This was followed by a continuous increase to 153% after 8 h of incubation. Incubation with 4:1, cholesterol/PC liposomes resulted in increased intracellular calcium levels compared to control of from 126% to 230% after from 2 h to 8 h incubation. Statistical analysis indicated that most of these increases were significant. In an attempt to determine calcium uptake rate we analyzed the time-dependency of calcium uptake in the SMCs using 45Ca2+ at incubation times from 1 min to 240 min. Fig. i shows the time course of calcium uptake in the SMCs after pretreatment with or without liposomes for 8 h. The results indicated that the calcium uptake was directly proportional to the cholesterol content of liposomes. In 4:1, cholester,~l/PC liposomes-treated SMCs the intracellular calcium level was significantly higher than the control values from 2 min to 240 min. in 1:1, cholesterol/PC liposomestreated SMCs the intracellular calcium level was significantly higher than control values only from 2 min to 5 rain of incubation. Using 4~Ca:* we estimated the kinetic parameters for the calcium uptake for linear uptake periods (the first 5 mint and the curvilinear uptake periods. The effect of cholesterol on the accumulation of intracellular calcium, summarized in Table It, was observed in both fast phase (linear uptake periods) and slow phase (curvilinear uptake). In the fast phase, l : l and 4:1, cholesterol/PC liposomes increased the pool size by about 1.6-fold and 1.7-fold compared with control values, respectively, and had no effect on rate constant. Therefore, influx (which equals rate constant × pool

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3

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o

I

120

60

I

I

180

240

Time ( r a i n )

Fig.l. Calcium uptake by the SMCs from ,1 min incubation with 45Ca2+ to 240 min at 37°C after pretreatment with 1:1 (o) and 4:1 (o), cholesterol/PC liposomes or without liposomes ( • ) . Each point represent the mean ± S.E. of three experiment performed in duplicate.

size) was increased by the same factor as pool size. In the slow phase, l : l and 4:1, cholesterol/PC lipos o m e s c a u s e d an i n c r e a s e in pool size o f a b o u t 1.0-fold a n d 1.9-fold, respectively, a n d t h e v a l u e s o f t h e r a t e c o n s t a n t s did n o t c h a n g e . T h e r e s u l t s s h o w e d t h a t for t h e slow p h a s e t h e e n h a n c e m e n t o f c a l c i u m influx results f r o m t h e i n c r e a s e o f pool size. T h e fast p h a s e p a r a m e t e r s w e r e m o r e sensitive to t h e i n c r e a s e o f c e l l u l a r c h o l e s t e r o l t h a n t h e slow p h a s e in t h e cells p r e t r e a t e d with 1 : 1 c h o l e s t e r o l / P C l i p o s o m e s . In o r d e r to o b s e r v e t h e e f f e c t o f c h o l e s t e r o l o n t h e activity o f ( C a 2 + + M g 2 + ) - A T P a s e in S M C p l a s m a m e m b r a n e s a n e x p e r i m e n t w a s c a r r i e d o u t in w h i c h t h e membrane fraction was isolated after incubation of the

TABLE 11

The effect of cholesterol in liposomes on (Ca 2 + + Mg 2 +)-ATPase in the SMCs

The cells were incubated with I : 1 and 4:1, cholesterol/PC (M/M) liposomes for 2 and 8 h at 37°C. Values are given as mean±S.E. and means within the same line with a superscript letter are statistically different (P < 0.05). Incubation n time (h) 2 8

Control

/* Mol Pi/mg pro. I : l cholesterol/ PC

9 9.99+0.36 9.83+0.44 9 9.01+ 1.41" 7.41+0.64

4: ! cholesterol/ PC 9.80+0.82 6.79±0.89 a

TABLE IV

The effect of liposomes ( l : l and 4:1, cholesterol~PC, calcium inflttr in the SMCs incubated for 8 It at 37°C

Control

M / M ) on

1:1 Cholesterol/PC

4:1 Cholesterol/PC

1 0.69 6.49 447

I O.69 10.10 6.97

1 0.69 10.97 7.57

Slow phase Half-time (min) 29 Rate constant train- 1) 0.02 Pool size (pmol/mg I,rot.) 5.15 Flux (pmol/mg prot. min) 0.12

29 0.02 5.23 0.13

29 0.02 9.62 0.23

Fast phase Half-time (rain) Rate constant (min - i ) Pool size (pmol/mg prof.) Flux(pmol/mgprot. min)

TABLE !11

Change in the SMCs membrane cholesterol and phospholipid (PL) content after an 8 h incubation with liposomes (1:1 and 4:1, choles. terol / PC, M / M) at 37°C

Data represent means in 10 independent experiments. Means within the same column with a superscript letter in common are statistically different (P < 0.05) Cholesterol (n tool/ mg prot.) Control 1 : 1, Cholesterol/PC 4 : 1, Choles. terol/PC

PL(nmol/ mg prof.)

Cholesterol /PL

Increase of cholesterol/ PL(%)

16.13 =

50.40

0.32

19.76

54.75

0.36

113

25.57 =

55.96

0.46

143

TABLE V Percentage phospholipid composition of the SMCs incubated with and without liposomes (1:1 and 4:1, cholesterol~PC, M / M) for 8 h at 37oc

Results are ~xpressedas mean± S.E. of five separated preparations and tool% values are given for the phospholipidcomposition Phospholipid

Control

Mol% l:l, choles- 4:l.cholesterol/PC terol/PC 52.81+ 1.80 54.24+ 1.41 55.59±1.60

Phosphatidylcholine Phosphatidylethanolamine Cardiolipin Sphingomyelin Phosphatidylinositol P~'osphatidylserine Pho:m~,atidic acid

25.43+0.78 23.26+1.19 23.84±0.64 2.42± 0.68 1.53± 0.23 1.31± 0.15 4.26+0,35 7.00±1.34 7.65+1.21 8.58± 0.14 6.23± 0.52 5.03± 0.59 1.97+ 1.09 2.47+ 1.66 1.95+ 1.19 6.06+ 0.42 7.54± 0.48 6.61 _+0.35

cells with liposomes. Comparison of control values to those obtained in the presence of liposomes at the same incubation times gave the results shown in Table lII. During incubation of the ceils with liposomes, the enzyme activity showed a decrease with both time and the cholesterol dose in liposomes. The maximal reductions were 17.7% and 24.6% ( P < 0.05) of the control value which were observed after 8 h of incubation in the m e m b r a n e s treated with i : l and 4 : 1, cholesterol/PC liposomes, respectively. The effect of an 8 h incubation of the SMCs with liposomes on the molar ratio of eholesterol/PL in membrane is shown in Table IV. The SMCs used in these experiments contained a molar ratio of cholesterol/PL of 0.32. After the cells were incubated with liposomes, this ratio in the cell membranes was increased by an amount directly proportional to the cholesterol content of liposomes. The cholesterol/PL ratios of the cells treated with 1 : ! and 4: 1, cholesterol/PC liposomes were 113% and 143% of control, respectively. Membrane PL are known to alter the activity of purified Ca2+-ATPase from human erythrocytes. Specifically, phosphatidylinositol (PI), cardiolipin (CL), phosphatidic acid (PA) and phosphatidylserin¢ (PS) induce a 3- to 5-fold stimulation of the enzyme [25]. Table V lists the PL composition of the normal and the liposomes-treated membranes. Although phosphatidylethanolamine (PE), PI and e L decreased and sphingomyelin (Sph) and PC increased, these changes were not significant. Discussion

Our results indicated that the increase in intracellular calcium in arterial SMCs was strongly influenced by increasing the cholesterol content of the cells

through liposome fusion. This accumulation was both cholesterol dose and incubation time-dependent. The observation that arterial SMCs are sensitive to the cholesterol concentration in the media is similar to results acquired using other membranes [14,26-28]. The correlation with cholesterol and increased intracellular calcium is consistent with other studies [13,14]. The conclusions of different investigators on the possible effect of cholesterol on calcium accumulation by the cells, however are not in agreement. The accumulation of intracellular calcium induced by cholesterol has been attributed to either stimulation of the Ca-'* channel in the membrane [13] or alteration of membrane fluidity [14]. However, we found that cholesterol treatment not only promoted 45Ca2+ influx, but also reduced the (Ca2++ Mg 2+)-ATPase activity in the membrane. We suggest that the accumulation of calcium induced by varying the cholesterol content in liposomes was achieved by an increase in calcium influx as well as a decrease of pumping calcium out. There are at least three possible effects of cholesterol responsible for the increase of intracellular calcium. First, the increase in the cholesterol/phospholipid ratio in the membrane can influence either the enzyme activity or Ca 2+ channel indirectly by modification of membrane fluidity, lnbar and Shinitzky [19] showed that fymphoma cells treated with lecithin-cholesterol lipo~omes increased the rigidity of the lipid layer. This increased rigidity resulted from the addition of cholesterol to a lipid bilayer resulting in a limitation of molecular motional freedom in the membrane [7], or from the effects of its rigid sterol structure on the other lipid components of the membrane leading to an increase in anisotropic motional order of the lipid bilayer [12]. This general increase in ordering may affect the eonformational transitions of the membrane-bound enzyme. Normal conformational transitions are necessary for most enzymes to perform ,their functions. Such an effect has been observed and e~

The effect of cholesterol on the accumulation of intracellular calcium.

Cholesterol/egg phosphatidylcholine (PC) liposomes (1:1 or 4:1, M/M), in which the absolute amount of PC was adjusted to be the same, were incubated w...
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