wllail#n(l221)12,

427470

~LongmmnCiroupUKLtd1221

The effect of 2Shydroxycholesterol on accumulation of intracellular calcium Q. ZHOU, S. JIMI, T.L. SMITH and F.A. KUMMEROW Burnsides Research Laboratory, Department of Food Science, University of Illinois, Urbana, Illinois, USA Abstract - Liposomes prepared with 2Shydroxycholesterol and egg phosphatldylchollne (PC) were incubated with bovine arterial smooth muscle cells for 8 h at 37-C. Cells incubated In the absence of llposomes or with liposomes containing cholesterol and PC were used as controls. The results indicated that calcium accumulated In the smooth muscle cells incubated in the presence of 2ShydroxycholesteroI containing llposomes In an amount proportional to the time of incubation. The calcium accumulationhas indicated by kinetic analysis, resufted from an increased compartment size. (Ca ++Mg2$ATPase exhibited decreased activity after pretreatment with 25-hydroxycholesterol containing iiposomes and the increased intracellular calcium content was dkqotly proportional to the decreased (Ca2++Mg2+)-ATPase activity. When lipids in the ceil membrane were examlned, a failure to change the cholesterol/phospholiplds ratio in the membrane was noted. The 25-hydroxycholesterol content in the membrane determined by HPLC dld not increase. An increase in sphlngomyelin and a decrease in phosphatidylethanolamlne and acldlc phosphollplds in the membrane was noted. We suggest ti the accumulatfon of intracellular calcium comes from both an increase of calciurnh~flux and a decrease of (Ca2++Mg2+)-ATPase activity, which may be the consequenqe .of changes In membrane phospholipld composition.

The aorta of chicks fed 7-ketocholesterol contains a significantly greater number of dead and dying smooth muscle cells than that of chicks fed only a basal diet [l]. 25Hydroxyvitamin D perturbs the arterial wall sufficiently to initiate intimal thickening in the coronary arteries of swine [2]. In rabbit it was shown that oxidized derivatives of cholesterol cause cell degeneration and induce focal intimal edema [3]. These morphological studies indicate that auto-oxidation products of cholesterol are injurious to the arterial wall. Consequently, they may play a role in atherogenesis although the

mechanism of oxidized sterols in atherogenesis is unclear. 2SHydroxycholesterol is an auto-oxidation product of cholesterol and may arise during the processing or cooking of cholesterol containing foods. 25Hydroxycholesterol may also be formed enzymatically in vivo [4]. It has been demonstrated that 2%hydroxycholesteroldepresses stem1 synthesis by inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase 15-71. The depressed enzyme activity results in a decrease in both cholesterol synthesis [Jl and the molar ratio of

467

468

cholesterol/phospholipids in the cell membrane 161 resulting in an alteration of biological activity including inhibition of DNA synthesis [7], depression of cell growth [8], change in cell shape 191, decreased rate of endocytosis [lo], increase in the fragility of cultured L cells [6 , and alteration in permeability to ‘$b+ [ 111and 44Ca” uptake [7]. There at least three factors involved in the maintenance of calcium gradients which are likely to be affected by membrane structure and composition. These am: (a) the calcium influx into the cell, mainly through a Ca2+channel in the membrane; (b) the removal of calcium from the cell by the pumping action of enzyme coupled reactions; aud (c) the Na+/Ca2+ exchange occurring mainly in the excitable cell membrane, and possibly sequestration into intracellular organelles and binding to proteins in the cytosol. Boissonneault and Heiniger [7, 121 demonstrated that 25hydrox~cholesterol induces an elevation in intracellular Ca2+. This elevated uptake of Ca2+ stems from HMG-CoA reductase inhibition leading to reduced removal from the cell by calcium pumps and to membrane permeability changes due to 25-hydroxycholesterol insertion into the membrane. The aim of the present study was to investigate the effect of 25hydroxycholesterol on intracellular calcium content and on membrane lipid composition in SMCs since the phospholi id content of the membrane affects (Ca2’+Mg!t )-ATPase activity [13-151 and membrane permeability 1161.

Materials and Methods Chemicals

Egg PC was obtained from Avanti Polar Lipid Inc., and cholesterol and 25-hydroxycholesterol from Sigma Chemical Co. All lipids were stored under nitrogen at -2o’C and were used without further purification. Pm&YAM was obtained from Molecular Probes Inc. Stock solutions of 1 mM Pura-2/AM were prepared with chloroform, dried under nitrogen and stored at -20°C. Working solutions were prepared immediately before use by diluting stored Pm&!/AM in dimethylsulfoxide (DMSO) and then by dilution to 1.5 pM in a balanced salt solution

cl3LL#-

(BSS). The BSS contained 20 mM HBPES buffered to pH 7.4 with Tris base, 135 mM NaCl, 5 mM KCl, 1 mM MgCl2,l mM CaC&, 10 mM ucose and 0.025% bovine serum albumin. 45Ca9+ was purchased from Amersham Co., diluted to 1 i/5 pl with deionized water and stored at 4°C. 41°C Ca2+ was added to Eagle’s Minimum Essential Medium (MEM: Gibco, Grand Island, NY, USA) to yield a working solution of 1 uCYO.5ml. Cell culture

Bovine arterial smooth muscle cells (SMCs) were prepared by the method of Jimi et al. [17]. SMC8 used in this study were in the 15th passage. The cells were grown to confluence in MBM supplemented with 20% fetal bovine serum (PBS: Sigma) at 37°C in a water-saturatedatmosphere of 5% COZ in air in 75 cm2 tissue culture flasks (Coming). The cells were harvested by using a trypsin-EDTA solution (Sigma) and transferred to flasks and 24well plates (Coming). Liposomepreparation

Liposomes were prepared by the method in Inbar and Sbinitzky [18] with modifications. Solutions of 40 mg of PC mixed with 20 mg cholesterol or 20 mg 25-hydroxycholesterol (l:l, M/M) in 2:l (v/v) chloroform/methanol were evaporated to dryness under nitrogen and dispersed in 26 ml of deionized water. The dispersions were then subjected to bath ultrasonic irradiation (Bransonic 220, Smithkline Co.) for 40 min in ice cold water. The sonicated solutions were centrifuged at 21000 g for 30 min at 4°C . The supematants which contained about 50% of the total lipids were collected, kept at 4’C and used on the same day. Just prior to use, 3.3-fold concentrated MBM was added to the suspension, adjusting it to the standard ion concentration of MBM (pH = 7.2). Incubationof the cells with liposomes

Confluent monolayer cultures of SMCs in flasks or 24well plates were washed three times with MEM without PBS. MBM, with or without liposomes, was added to the SMCs in the flasks (8 ml) and in

25-HYDROXYCHOLESV3ROLz INTRACELLULAR CALCIUM ACCUMUL,ATION

the 24well plates (1 ml), and the cells were incubated up to 8 h at 37°C. The viability of the SMCs, as determined by dye exclusion, was not significantly altered after an 8 h incubation with or without liposomes. Measurementof [Ca2’]i withFura-2iA.M

After incubation of the cells with liposomes for 2,4, 6, or 8 h, the vesicle suspensions were aspirated from the culture flasks and the cells were washed three times with 8 ml of MEM. Then, 8 ml of 1.5 pM Fura-2/AM was added to each tlask, and the culture was incubated at 37-C for 60 min. The medium was removed from the flasks and the culture washed three times with 8 ml of BSS. The cells were removed from the flasks by incubation with trypsin (Sigma) for 1 min followed by scraping with a rubber policeman. They were then washed three times with 50 ml cold BSS and then centrifuged at 250 g for 3 min after each washing to remove trypsin and extracellular Fur&?/AM. The cells were then suspended in 7.5 ml of BSS on ice and triplicate 2.5 ml aliquots of cell suspension were placed in 1 cm quartz cuvettes for measurement of [Ca2+]i with continuous stirring at room temperature. The samples were excited alternately at 340 and 380 nm (bandwidth = 5 run) and fluorescent emission intensity was monitored continuously at 510 run (bandwidth = 5 run). Fmaxand Fti were obtained by the addition of 8 pl of 100% Triton X-100 and 150 pl of 400 mM EGTA, respectively. [Ca2+]iwas calculated from the fluorescence signals as described by Grynkiewicz et al. [19]. Autofluorescence was determined in samples of the cells treated with 15 pl DMSO without Fur&/AM. No leakage of the dye outside the cells was noted as evaluated by addition of 1 mM Mn2’ to the cell suspension just before recording the spectra. Measurementof45C2+ influx

The confluent cells seeded in 24well plates were gently rinsed three times with MEM at 37°C after being pm-incubated with or without liposomes for 8 h. After washing, 0.5 ml of 45Ca2t solution (1 PC) was added to the wells, and the cells were incubated at 37°C for time intervals from 1 min to 240 min.

46B

the radioactive medium was removed, the monolayers were washed three tunes with cold 0.1% phosphate buffered saline (PBS) containing 1 mM EGTA and digested in 1 ml of 0.5 N NaOH. Then 0.8 ml of the digested cells were counted in a liquid scintillation counter and 0.2 ml was assayed for protein (Bio-Bad Co.). The kinetic parameters for the calcium influx were calculated by the method of Borle 1203. After

Measurementof lipiak

The procedure of Pikul et al. [21] for tissue lipid extraction and measurement was used with modifications. The cells used for the measurement of lipids were pretreated for 8 h with or without liposomes, washed five times with PBS, harvested by brief trypsin treatment and washed tsvice with 15 ml of PBS with centrifugation at 600 g, followed by 15000 g for 10 min at 4°C. The cell pellets were 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) at 4’C overnight Separation of cholesterol and phospholipids (PL) from the lipid extract was accomplished by TLC using petroleum etherkiiethyl ether/glacial acetic acid (80:20:1, v/v/v). Separation of phospholipids was achieved by two dimensional TLC, using chloroform/methanol./15N ammonium hydroxide (65:25:5, v/v/v) and chloroformketon~ methanol/glacial acetic acid/water (3:4:1:1:0.5, v/v/v/v/v) according to Nelson et al. [22]. Total cholesterol and phophoms (Pi) in the extract were assayed [211. The molar ratio of cholesterol/PL and the molar percentage of PL were calculated The total 25-hydroxycholesterol in the cells was analysed by HPLC according to Kou et al. [23]. Assay of (Ca2++Mg2+)-ATPase Pre{.ratio;+of SMC membranes to be assayed for (Ca +Mg )-ATPase was amended from the procedure of MOOR 1241. The SMC monolayers were washed three times with PBS after incubation at 37’C with or without liposomes and harvested by trypsinixation. The fragmentation of the membrane wasperformedbywashingthreetimeswith15ml of 15 mM Tris buffer @H 7.4) containing 1 mM

470

cELLc!ALnuM

Table 1 Qmntitation by Fura- of f?ec intracellular caloium in SMCs incubated with cholc&e&PC or 25hydroxycholesteml(2S-OHCHOL)PC liposomea (X:1, M/M) for 2.4.6, and 8 h at 37X. baa&at&n

[CU2+]i (n

(h)

time

n

2

Control

17

1048f466blc * *

,

MOUml)

choleAterouPc

25-OHCHOLIPC

121.6 f 5Mbl,b2

167.0 f 5.59%

4

8

101.8 f 6.88a

122.2 f 7.86

166.1 f 16.5”

6

10

108.5 f 6.21a*b

158.7 i 19.F

174.0 -I 17.5b

8

13

105.2 f 5Mb

161.1 f 24.e

267.2 f 30.pb’

Values am given as means f SE. Means within the same line with a superscript letter in common are stati&Aly letter of a, b, c represent statistical differences at levels of P < 0.05, P < 0.01 and P < 0.001 respectively

diffenmt and the

EDTA (neutralized to pH 7.0 with NaOH) followed The results were tested using a multiple analysis by centrifugation at 15000 g for 10 min, then by of variance and the Scheffe test. washing twice with 10 mM Tris buffer (pH 7.4) in the same manner. One ml of deionized water was added, and duplicate 50 pl aliquots of the R&QlltS suspension were used for measurement of protein concentration; 900 pl of suspension was used to The addition of liposomes containing measure (Ca2’+Mg2+)-ATPasein triplicate, follow- 25-hydroxycholesterol to the media evoked a ing the method described by Moom et al. [241. striking increase in the free intracelhrlar calcium of Activities were expressed as pmol Pi/mg membrane SMCs, as shown in Table 1. The basal level of intracellular calcium fluctuated between 101.8 nM protein.

a 10

t

F Qi

5

t3

Q

Qx

x

1

0

Fig.1 The uptake

6 x

x

60

x

120

lime(min)

180

246

of calcium from 1 min incubation with %a2+ to 240 min at 37’C after pretreatment with cholestemUFC (open circles)

and 25-hydroxychole&erol/W

(fllled circles) liposomes (1:l. MM) or witbout liposomes (open triangles). Tbe vahus nprrrsat

of three experiments pe&bnned in duplicate

means

25-HYDROXYCHOLESTEROL:

INTRACELLULAR

471

CALCIUM ACCUMULATION

Table 2 Ihe effect of 8

h incubation with cholesterol/FC or 25-hydroxycholesterol(25-OHCHOLYPC lip~omes (1: 1, M/M) on calcium influx and pool size in SMCs Parameter

Control

cholestffolJPc

25OHCHOLIPC

Fast phase

Half-time (min) Rate constants (ruin-‘) l%x@ mol/mg prot min) Compartment size (p mol/mg prot)

1

1

1

0.69

0.69

0.69

4.47 6.49

6.97 10.10

9.16 13.28

29

29

Slow phase

Half-time (min) Rate constants (min?) Flux@ mol/mg prot min) Compartment size (p mol/mg prot)

29

and 108.5 nM during an 8 h period. After incubation of the cells with 25-hydroxycholesterol or cholesterol, there was a time-dependent increase in free calcium content. The intracellular calcium of 25hydroxycholesterol incubated cells increased from 167 nM after a 2 h incubation to 267 nM after an 8 h treatment. The cells treated with cholesterol containing liposomes also showed an increase in fret intracellular calcium. However, this increase was less than that produced by 2%hydroxycholesterol liposomes during the entire period of measurement Statistical analysis indicated that the free calcium content increase by 25-hydroxycholesterol was significantly higher than liposomefree controls and cholesterol-treatedcells. We then investigated the effect of 25-hydroxycholesterol on calcium influx into SMCs using 45Ca2’at incubation times from 1 min to 240 min. Figure 1 shows calcium uptake values measured with or without treatment with liposomes for 8 h. Calcium uptake deviated from linearity after the first 5 min of incubation and then exhibited curvilinear behaviour. The results show that incubation with 25-hydroxycholesterol containing liposomes evoked a si nificant increase in the uptake of cellular 45Cafi+ after 2 min when compared with the liposome free control group and after 3 min when compared with the cholesterol treated group. From 2 min to 5 min the cholesterol treated SMCs also had significantly greater 45Ca2t uptake in comparison with the control group,

0.02

0.02

0.02

0.12 5.15

0.13 5.23

0.18 7.53

Based on the uptake of 45Ca2’,we estimated the kinetic parameters of the calcium uptake. As shown in Table 2, the treatment of cells with 25-hydroxycholesterol containing liposomes led to an increased compartment size in both the fast phase and slow phase and no change in rate constant In the fast phase, 25-hydroxycholesterol liposomes increased compartment size about 2-fold while cholesterol increased only 1.6-fold. Infhrx (which equals rate constant times compartment size) was increased by the same factor as compartment size. In the slow phase, 25-hydroxycholesterol caused an increase in compartment size of about 1.5-fold, but cholesterol had no observable effect on compartment size. To assess the influence of 2%hydroxycholesterol on the activity of (Ca2++Mg2t)-ATPase,constant concentrations of 25-hydroxycholest or cholesterol in liposomes were added. Table 3 shows that, in the absence of sterol, (Ca2++Mp)-ATPase activity was found to be 9.99 pm01 Pi/mg protein after a 2h incubation, followed by a decrease to 7.09 pm01Pi/mg protein after a 4 h incubation then gradually recovering to 9.01 pm01Pi/mg protein after an 8 h incubation. In the presence of 25-hydroxycholesterol containing liposomes, a depression of enzyme activity occurred over the entire period of measurement The decrease was significant when compared with the control value at the same incubation time. The treatment of the cells with cholesterol containing liposomes affected this parameter only slightly. In addition, a negative

472

CELLcXLCNh4

Table 3 The effect of cholesterol/PC OT2%hydroxycholesterol(25OHCHOL)/PC (Ca2++Mg2+)-ATPase activity in SMCs

lipmomes (1: 1. M/M) on

pnwl PJmg protein Time (h)

n

Control

choksterouPc

2 4

25-OHCHOWPC

9

9.99 f 0.3@

9.83 f 0.44

8

7.09 f 0.4T

6.98 f 0.22

5.83 f 0.35*

6

8

7.88 f 1.18*

7.65 f 1.59

5.65 f 0.6s”

8

9

9.01 f 1.41’

7.41 f 0.64

5.65 f 0.6a’

8.41 f 0.56

Tk cells were incubated with the liuosomes for 2,4,6 and 8 h at 37°C.Meanswithin the same line with a superscriptletter am statistically difkent (P < 0.05) comlationbetw~ntheaccumulation ofintracellular calcium and (Ca2++Mg2+)-ATPase activity in membranes was demonstrated over the entire period in the 25hydroxycholesterol treated cells. That is, the content of calcium was progressively increased while there was a gradual decrease in the enzyme activity. The maximal 2.5-fold increase of free intracellular calcium was accompanied by a maximal decrease of 37.3% in the enzyme activity after an 8 h incubation. The analysis of the effect of 25hydroxycholesterol on membrane lipids showed a failure to change cholesterol content in the membrane after an 8 h incubation of the SMCs with W-hydroxycholesterol containing liposomes. The SMCs used in these experiments contained a molar ratio of 0.32 cholesterol/phospholipids. After the cells were incubated with cholesterol or 25hydroxycholesterol containing liposomes, the ratio in the cell

membranes was 0.36 and 0.31 respectively. Moreover, no difference in 25hydroxycholesterol content in the membrane was detected by HPLC between the normal and the liposome-treated cells. In order to observe the effect of 25hydroxycholesterol on the phospholipid content of the membrane, an analysis of the major membrane phospholipids was perfomred. Table 4 lists the phospholipid composition of the normal and the liposome treated membranes. After pm-treatment of the cells with 25-hydroxycholesterol containing liposomes, the phosphatidylethanolamine (PF9 was significantly decreased and the sphingomyelin (Sph) significantly increased. Although the change of acidic phospholipids, including phosphatidylinositol (PI), cardiolipin (CL), phosphatidylserine (PS) and phosphatidic acid (PA), was not significant, there was a decreasing trend in the content of these phospholipid components in the membrane.

Table 4 Percentage phospholipid composition of smooth muscle cells incubated with liposomes containing cholesterol/PC or 25-hydroxycholest (25-OHCHOL)/PC (1: 1, M/M), for 8 h at 37-C Phospholipid

Control

cholesterouPc

25-OHCHOLIPC

Phosphatidylcholine

52.81 f 1.80

54.24 f 1.41

56.57 f 1.34

Phosphatidylethanolamine

25.43 f 0.78”

23.26 f 1.19

21.11 f 0.72’

Cardiolipin

2.42 f 0.68

1.53 f 0.23

1.49 f 0.16

Sphingomyelin

4.26 f 0.35’

7.00 f 1.34

9.85 f 1.32’

Phosphatidylinositol

8.58 f 0.14

6.23 f 0.52

4.25 f 1.03

Phosphatidylserine

1.97 f 1.09

2.47 f 1.66

1.38 f 0.84

Phosphatidic acid

6.06 f 0.42

7.54 f 0.48

5.27 f 1.34

are expressed an mean f SE of mol% values fxumfive separatepnparations and a supersu-iptletter in commcn is statisticflllydBen%lt(P < 0.05)

Redts

25HYDROXYCHOLESTEROL:

INTRACELLULAR

CALCIUM ACCIMULATION

Discussion A previous study has shown that the exposure of P815 murine mastocytoma cells to 25hydrox 1+ cholesterol induces an increased rate of 45Ca uptake into the cells due to an inhibition of HMGCoA ieductase [7]. The reduced production of cholesterol is believed to be res nsible for the go altered membrane permeability to ‘Ca2’ [5-7, 121 through reduced content of membrane cholesterol. This study, however, was performed in tumor cells during the rapid proliferation period [7] when cells need to synthesize more cholesterol in order to produce new membrane. In quiescent periods, cholesterol is only synthesized to meet metabolic An inhibiting effect of 25hydroxydemands. cholesterol on HMG-CoA reductase in proliferating tumor cells should be much more serious than in the quiescent or more slowly growing normal cells. The possibility exists that the inhibiting effect of 25-hydroxycholesterol on HMG-CoA reductase is not an important factor in increased intracellular calcium content of quiescent cells exposed to 25-hydmxycholesteml. We did not determine HMG-CoA mductase activity. The fact that the cholesterol content of the membrane did not decrease supports our view that the inhibition of HMG-CoA reductase was not responsible for the increased intracellular calcium under our test conditions. The fact that mevinolin, a potent competitive inhibitor of HMG-CoA reductase, did not alter 45Ca2+ uptake by P815 cells supports our contention that there are other effects of 25hydroxycholestero1, unrelated to its known effect on HMG-CoA reductase [12]. Oxidized sterols are normally carried through the blood in lipoproteins and consequently enter the cell by endocytosis. The liposomes used in this study provide a more physiologically relevant means of administering the 25-hydroxycholesterol than has previously been used. These liposomes am unable to fuse with the cell but are taken up by non-specific endocytosis. At this point we cannot determine if this method of administration of 25-hydroxycholesterol may be responsible for the assumed lack of effect on HMG-CoA mductase. There is evidence that oxidized stem1 molecules can insert into and disrupt the lipid bilayer [25].

413

The incorporation of 0.5 mol% 25-hydroxycholesterol into liposomes substantially increases the permeability of liposomes to Ca2’ 1261. Cholesterol present at 10 mol% resulted in liposomes which are impermeant [26]. This indicates that only minute quantities of oxidized stem1 are necessary to perturb membranes. It is, therefore, possible that inserted 25-hydroxycholesterol plays a role in perturbation of membrane [26]. Such a hypothesis would explain some of the observations both in *$bt transport in 25-hydroxycholesterol treated L-cells [ll] and in 45Ca ’ uptake into P815 cells [12]. The failure to detect increased 25-hydroxycholesterol in the cell membrane after incubation of the cells with 25-hydroxycholesterol containing liposomes is inconclusive. Since such small amounts of oxidized sterol are capable of causing a disruption in the membrane, the amount of 25-hydroxycholesterol present in the incubated cells may not be detectable by our HPLC detector. Shore et al. [27] report that phospholipids are synthesized by the arterial cell rather than derived from the plasma. M&&less and Zilversmit [28] demonstrated further that Sph increases most significantly in phospholipids of the atheromatous plaque of the cholesterol-fed rabbit, Similarly, a relative increase in Sph concentration in human atheromatous aorta and coronary vessels has been observed 1291. We here observed that the effect of 25-hydroxycholesterol on Sph synthesis is much stronger than that of cholesterol, although the mechanism by which 25-hydroxycholesterol caused changes in phospholipid composition is unknown. These changes in phospholipid composition are in good agreement with previous results which show that phospholipid proportions in membranes are closely related to intracelhrlar calcium content 113-16, 30-341. So we propose that an increase in Sph synthesis, mediated by 25-hydroxycholesterol, may enhance calcium influx. Support for this hypothesis comes from the results reported by Yla-Herttuala et al. [30]. They found that in a high coronary heart disease risk population, the deposition of caIcium in the coronary arteries was directly proportional to an elevated Sph. The increase in Sph in the cells treated with 25-hydroxycholesterol is accompanied by a decrease in PE. This is not an expected result, since Sph is

474

synthesized from PC and an increase in Sph would be expected to be accompanied by a decrease in PC rather than PE. There exists, however, a minor pathway that directly converts PE to PC [35]. This pathway is thought to be significantly active only in the liver, but if it is, or were to become, active in these SMCs, the net result could be the same as the change shown in Table 4. The precise method by which these enzymes may be influenced remains to be determined. We suggest that Sph may increase Ca2+ influx mainly by increased calcium bonding to the cell membrane. Sph located on the exterior of the plasma membrane [36] has an exposed polar head group accessible to the aqueous environment. The negative charge on Sph would thus be accessible for ionic bonding with Ca2+. This is supported by our finding that the compartment size in the fast phase was increased. BoissoMeault and Heiniger [12] ;;gg;f that 25hydroxycholest stimulates Ca influx into the cell in a manner that is dependent on bringing calcium to extracellular receptors. Calcium channel blockers, however, had no effect on 45Ca2t influx into 25-hydroxycholesterol treated cells [12] and the rate constants showed no change in our experiment. These observations indicate that the influence of 25hydroxycholesterol on 45Ca2+influx in SMCs resulted mainly from the effect of oxidized sterols on compartment size rather than from its effect on the calcium channel. The alteration of extracellular compartment size may be under the influence of Sph content as discussed earlier. Respiratory inhibitors, DNP, NaN3 and NaCN, can depress the slow phase accumulation of 45Ca2+induced by 25-hydroxycholesterol [12], indicating that the effect of 25-hydroxycholesterol is related to mitochontia. Due to experimental design, we were unable to distinguish between plasma membrane and mitochondrial effects. The majority of PE and PS are located on the inside of the plasma membrane [36], which allows access to (Ca2++Mg2+)-ATPase. They are also major components of the mitochondrlal membrane. PE appears to stimulate activity of the calcium pump protein [14-16, 311. Euhanced PE content in mcoustituted membraues leads to an increase in calcium pump activity [14] while a lowering of PE

cELLcTALcxM

content in sarcoplasmic reticulum was accompanied by a decrease in pump activity [lS]. These studies imply a role for PE on calcium pump function in native membraues. In the light of these observations and our observation that PE content decreased significantly, we propose that 25-hydroxycholesterol enhancement of intracellular calcium concentration was partly due to a decrease in PE which leads to a reduction in (Ca2t+Mg2t)-ATPase activity. In addition to the effect of PE, there is evidence that acidic phospholipids are capable of activating calcium transport. purified (Ca2’+Mg2’>ATPase from erythrocyte membrane can be activated in both a solubilized and a reconstituted system bl the addition of acidic phospholipids [16]. The (Ca ++Mga+)-ATPaseactivity was lost after removal of the phospholipids in erythmcyte ghosts using phospholipase A2, whereas phospholipiddepleted (Ca2++Mg2+)-ATPasecan be reactivated by addition of PS [32]. Au increase in the percentage of PS in liposomes enhances the activity of the enzyme when reconstituted into liposomes [16]. This elevated activity results from PS imitating the effect of calmodulin on the isolated (Ca2’+Mp>ATPase [33]. These studies [16, 32, 331 indicate that these agents appear to act directly on the purified enzyme rather than on membrane modification [15]. There is also evidence that PA cau act as a calcium ionophom [34, 371. An increase of PS*+PIor PE in liposomes substantially reduces the Ca permeability of the liposomes [26]. It seems that the effect of acidic phospholipids on the accumulation of intracellular calcium may be due to more than one factor. Conclusion We conclude that the effects of 25-hydroxycholesterol on calcium accumulation by bovine SMCs are multi-faceted. External binding of calcium may be enhanced by increased synthesis of Sph, the synthesis of which is stimulated by the oxysterol in an unknown manner. Removal of intracellular calcium by ATPase is reduced, possibly due to the loss of PE. In quiescent cells the inhibition of HMG-CoA mductase by the oxysteml appears to have a minimal effect, but this issue remains to be clarified.

25-HYDROXYCHOLESTERO~ INTRACELLULAR CALCIUM ACCUMULATION

Acknowledgements This

work

was

supported

by

the

Wallace

Genetic

15.

Foundation. 16.

References 1. Toda T. Lesxczynski DE. Kummerow FA. (1982) Angiotoxic etfects of dietary ‘I-ketocholesterol ill chick aorta. Arterial Wall, 7, 167-176. 2. Holmes RP. Kummetow FA. (1983) The relationship of adequate and excessive intake of vitamin D to health and disease. J. Am. Coll. Nutr., 2, 173-199. 3. Jmai H. Werthessen NT. Taylor CB. Lee KT. (1965) Angiotoxicity and arteriosclerosis due to contaminants of USP-grade cholesterol. Arch. Pathol. Lab. Med., 100, 565-573. 4. Jmai H. Werthessen NT. Subramanyam Y. Kanisawa M. (1980) Angiotoxicity of oxygenated sterols and possible pmcursors. Science, 207,651-653. 5. Kandutsch AA. Chen HW. (1974) Inhibition of stem1 synthesis in cultured cells by cholesterol derivatives oxygenated in the side chain. J. Biol. Chem., 249, 60576061. 6. Kandutsch AA. Chen HW. (1977) Consequences of blocked stem1 synthesis in cultured cells. J. Biol. Chem., 252,409-415. 7. Boissomteauh GA. Heiniger H-J. (1984) 25-hydroxycholestcrol-induced elevation in “Ca uptake: correlation with depressed DNA synthesis. J. Cell Physiol., 120, 151-156. 8. Cox DC. Comai K. Goldstein AL. (1988) Effects of cholesterol and 25-hydroxycholesterol on smooth muscle cell and cndothelial cell growth. Lipids, 23,85-88. 9. Lin PS. Chen HW. (1979) Diminution of L-cell micmvilli following exposum to 25-hydmxycholestcrol. Cell Biol. Jnt. Rep., 3,51-59. 10. Heiniger H-J. Wolt JM. Chen HW. Meier H. (1979) A micro-method for lymphoblastic transformation of mouse lymphocytes from peripheral blood. Proc. Sot. Bxp. Biol. Med., 143, 6-11. 11. Chen NW. Heiniger H-J. Kandutsch AA. (1978) Alteration of *51b’ influx and efflux following depletion of membrane stem1 in L-cells. J. Biol. Chem., 253, 3180-3185. 12. Boissonneault GA. Heiniger H-J. (1985) 25-hydmxycholestcrol-induced elevation in “Ca uptake: permeability changes in P815 cells. J. Cell Physiol., 125, 471-475. 13. Navarro J. Toivio-Kinnucan M. Racker E. (1984) Effect of lipid composition on ti_calcium/adenosine 5’-triphosphate coupling ratio of the Ca”‘-ATPase of sarcoplasmic mticuhrm. Biochemistry, 23, 130-135. 14. Hidalgo C. Pettwci DA. Vergam C. (1982) Uncoupling of Ca2+lmnsport in samoplasmic reticulum as a result of labelling lipid ammo gmups and inhibition of Ca2+-ATPase

17.

18.

19.

20.

21.

22. 23.

24.

25.

26.

27.

28.

29.

30.

475

activity by modification of lyaine residues of the Ca”-ATPase polypeptide. I. Biol. Chem., 257.208-216. Niggli V. Adunyah Es. Car&Ii B. (1981) Acidic phosphohpids, unsaturated fitty acids, and limited proteolysis mimic the effect of calmodulin on the ptaified c+hrocyte Ca2+-ATPase. J. Biol. Cbsm.. 256, 8588-8552. Holmes RP. Yoss NL. (1984) U-h&dtoxystuols inuease the permeability of liposomes to Ca and other cations. Bicchim. Biophys. Acta, 770.15-21. Jimi S. Smith TL. KummerowFA. (1990) 25-Hydroxycholesterol-stimulated DNA synthesis iu smootb muscle cells and induction of endothelial injury using a coculture technique. B&hem. Med. Met. Biol. J., 44, 114-125. Jnbar M. Shinitxky M. (1974) hxxease of cholesterol level in the surface membrane of lymphoma cells and its inhibitory effect on ascites tumor development. Pmt. Natl. Acad. Sci. USA, 71,2128-2130. Grynkiewicz G. Poenie M. Tsien RY. (1985) A new generation of Ca2+indicators with greatly improved fluorescence properties. J. Biol. Chem., 260,3440-3450. Borle AB. (1975) Methods for assessing hormone effects on calcium fluxes in vitro. In: Hardman JG.O’Malley BW. ed. Methods in Enzymology, Hormone Action. Vo139. New York, Academic Press, pp. 513-573. Pikul J. Leszczynski DE. Kummero w FA. (1984) Relative role of phospholipids, triacylglycerols and cholesterol esters on malonaldehyde formation in fat extracted from chicken meat. J. Food. Sci., 49,704708. Nelson GJ. ed. (1972) In: Blood Lipids and Lipopmteins. New York, Wiley Interscience, pp. 45-50. Kou I. Holmes RP. (1985) The analysis of 25-hydroxycholestcrol in plasma and cholesterol-contaming foods by high performance liquid chromatography. J. Chmmatogr., 330, 339-346. Moore RB. Manery IF. Still J. Mankad VN. (1989) The inhibitory effects of polyoxyethylene detergents on human erythrocyte acetylcholinestemse and Ca*t@-AT&se. Biochem. Cell Biol., 67, 137-146. Hsu RC. Kanofsky JR. Yachnin S. (1981) The formation of echinccytes by the insertion of oxygenated stem1 compounds intc red cell membranes. Blood, 56, 109-l 17. Matsumoto T. Fontaine 0. Rasmussen H. (1981) Bffect of 1,25dihydroxyvitamin D3 on phospholipid metabolism in chick duodenal mucosal cell. J. Biol. Chem., 256, 3354-3360. Shore ML. Zilversmit DB. Ackerman RF. (1955) Plasma phospholipid deposition and aortic phospholipid synthesis in experimental atherosclerosis. Am. J. Physiol., 181.527-531. McCandless EL. Zilversmit DB. (1956) The effect of cholesterol on the turnover of lecithin, cephalin and sphingomyelin in the rabbit. Arch. B&hem. Biophys., 62, 402-410. Steele JM. Kayden HJ. (1955) Nature of the phospholipids in human serum and athemmatous vessels. Trans. Ass. Am. Physic., 68.249-254. YlH-Herttuala S. Sumuvuori H. Karkola K. Mottonen M.

476

3 1. 32.

33.

34.

CELLcxLauM Nikkari T. (1987) Atbemsckrosis and biochemical composition of coronary arterks in Finnish mem comparison of two populations with di&ent incidences of conmary heart Athwosckrosis, 65, 109-115. KnowleaAF. RackerE. (1975) Proper& of a reconstituted calcium pump. J. Biol. Ckxu., 250,3538-3544. Roarer P. Qazzotti P. Car&h E. (1977) A lipid mquirement for the (Ca2++@>activated ATPase of etythmcyte membranes. Arch. Biochem. Biophys., 179, 578-583. Stieger J. Luterbacher S. (1981) Some properties of the purifii @x2++@)-ATPase from human red cell membranes. Biochim. Biophys. Acta, 641,270-275. Tyson CA. van de Zande H. Cheen DE. (1976) Phospholipids as ionophoms. J. Biol. Chem., 25 1, 1326-1332.

35. Vance DE. (1985) Phospholipid metabolism in leukoqks. In: Vance DE.Vance JE. ed. Biochemistry of Lipids and Membrane. Benjamin Cnmming Publishing Co, pp. 242-270. 36. van Deenen LLM. (1981) Topology and dynamks of phospbohpids in membranes. FEBS Lett., 123.3-15. 37. Ohsako S. Deguchi T. (1981) Stimulation by phosphatidic acid of cakium influx and cyclic 0MP synthesis in netuobkstoma cells. J. Biol. Chem., 256, 10945-10948. Please send reprint requests to : Dr Fred A. Rmmnerow, Bumsides Research Laboratory, University of Illinois, 1208 W. Pennsylvania Ave., Urbana, IL 61801, USA Received : 13 February 1991 Revised : 15 March 1991 Accepted : 6 May 1991