Inhibition of oxygenated sterol entry into human red cells and lymphocytes by isolated serum lipoproteins

538

Biochimica et Biophysics Ada, 713 (1982) 538-546 Elsevier Biomedical Press

BBA 51264

INHIBITION OF OXYGENATED LYMPHOCYTES BY ISOLATED STANLEY

YACHNfN

‘*‘, JIWHEY

CHUNG

STEROL ENTRY INTO HUMAN RED CELLS AND SERUM LIPOPROTEINS a and ANGELO

M. SCANU

Departments of’ Medicine and ’ 3iochemistr.y~ and ’ The Commitiee Chicago, IL 60637 (U.S.A.) (Received

a*b

on Immunologv,

University

of Chicago, 950 E. 59th St., Box 420,

June Ist, 1982)

Key words: Clxygenated sterol; StepoI uptake; Lipoprotein;

(Blood cet!)

We studied the uptake of certain oxygenated sterols by human lymphocytes during their incubation in lipoprotein-depleted medium and found that it resembles closely the uptake of oxygenated sterols by human red-cell membranes. Entry of oxygenated sterols into lymphocytes is virtually complete within 5 min of exposure of the cells to the oxygenated sterol-confining rn~i~, is related to the concen~ation of oxygenated sterols and is impeded by low temperature. The presence of free cholesterol does not alter the amount of oxygenated sterol entering lymphocytes. However, substitution of lipoprotein-containing medium for lipoprotein-depleted medium reduces the amount of oxygenated sterol entering lymphocytes by 65-75%. Oxygenated sterol uptake by lymphocytes from individuals with homozygous familial hypercholesterolemia does not differ from that of norma lymphocytes. Oxygenated sterols bind to all density classes of human li~proteins, but do not alter their hydr~ynamic properties of their cholesterokprotein mass ratios. These compounds also have little or no effect on cholesterol exchange between incubation medium and red-cell membranes. Both LDL and HDL, when added to oxygenated sterol-containing medium, effectively diminish the amount of oxygenated sterol taken up by red cells and lymphocytes; LDL is approximately 2.5-4 times more effective than HDL in preventing oxygenated sterol entry into cells. Lipoproteins can also act as acceptors of oxygenated sterols previously inserted into lymphocytes. Since oxygenated sterols have been reported to be atherogenie, the mutating effects of ii~proteins on oxygenated sterol uptake by cells, and the alterations in membrane structure and function which ensue, may be a useful model for further study.

Introduction

functions and on -membrane morphology. Based on a variety of experimental observations using the human red cell as a model, we have postulated that oxygenated sterols evoke these various membrane changes as a consequence of their physical insertion into the plasma membrane lipid bilayer [6,8]. In addition, utilizing lipoprotein-cont~ning and lipoprotein-deficient incubation media, we have shown that the insertion of oxygenated sterols into human red-cell membranes, as well as the consequent transformation of the red cells to an echinocyte shape and the expansion of the red-cell membrane, are impeded but not abolished by the

The exposure of mammalian cells to oxygenated sterol compounds in tissue culture produces marked metabolic changes affecting the sterol synthetic pathway [I]. In addition, we and others f2-91 have shown that oxygenated sterols have wide-ranging effects on cell membrane-associated

Abbreviations: VLDL, very low density lipoprotein(s); IDL, intermediate density lipoprotein(s); LDL, low density lipoprotein(s); HDL, high density lipoprotein(s).

mO5-2760/82/000%0000/$07..75

0 1982 Elsevier Biomedical

Press

539

presence of serum lipoproteins during exposure of red cells to the compounds [lo]. Since we also showed that all density classes of human serum lipoprotein bind oxygenated sterols, we concluded that lipoproteins interfere with oxygenated sterol membrane insertion by competing with cell membranes for the sterol compounds, thus rendering them unavailable for entry into cells. Furthermore, oxygenated sterols previously inserted into red cells can be removed by subsequent incubation in lipoprotein-containing medium, but not by exposure to other human serum proteins or bovine serum albumin. In the present report we extend our observations on the effects of human lipoproteins on the entry of oxygenated sterols into red-cell membranes using purified lipoprotein fractions. Furthermore, we have studied the characteristics of oxygenated sterol entry into nucleated human cells and its modulation by serum lipoproteins, in order to compare this process with that in red cells, and perhaps clarify what role oxygenated sterol uptake by nucleated cells plays in producing alterations in those membrane-associated functions not expressed by the red cell. Materials and Methods Cholesterol and 20a-hydroxycholesterol were from the Sigma Chemical Co. (St. Louis, MO). All other sterols were products of Steraloids, Inc. (Wilton, NH). [4-‘4C]Cholesterol (over 50 mCi/ mmol) was purchased from the Amersham Corporation (Arlington Heights, IL). Long-term human B-lymphocyte lines were obtained from the Institute for Medical Research (Camden, NJ). They were maintained in culture in 10% heat-inactivated fetal calf serum/90% RPM1 1640 (GIBCO, Grand Island, NY). Four different cell lines were employed in our studies; Nos. 130 and 946 were from normal adults, and Nos. 1458 and 1459 from individuals with homozygous familial hypercholesterolemia. Lines 1458 and 1459 were shown to be deficient in low density lipoprotein (LDL) receptors, by the method of Pittman et al. [ 111, and were also insensitive to inhibition of sterol synthesis from [ 14C]acetate precursor by exogenous LDL added to a lipoprotein-depleted incubation medium [ 121 (Yachnin, S., unpublished data). Prior

to experimental use the cells were washed three times in RPM1 1640. In studying the uptake of oxygenated sterols by the long-term lymphocyte cell lines (20-40). lo6 cells were incubated together with the desired concentration of sterol in 10 ml of RPM1 1640 supplemented with 20% (v/v) lipoprotein-depleted serum [8,13]. The sterols were dissolved in ethanol at 100-200 times their desired final concentration in culture and were added to 5 ml of lipoprotein-depleted medium, which in turn was added to 5 ml of the same medium containing the desired number of cells. This method was employed to reduce cell clumping, which sometimes occurred during incubation if the oxygenated sterol ethanol solutions were added directly to the cell suspension. Control cultures received equal volumes (0.5- 1.O% v/v) of ethanol. In some experiments heat-inactivated type AB human serum was used in place of lipoprotein-depleted serum. Both media contained equal amounts of protein (10 mg/ml), but lipoprotein-depleted medium had a cholesterol concentration of at most 4 pg/ml, while that of AB serum medium was 200 pg/ml. Where desired, various lipoprotein fractions were added to the cell suspension prior to addition of the oxygenated sterol solutions. The 10 ml cell suspensions were incubated in stationary 25-cm* tissue culture flasks (Falcon, Cat. No. 3013) at 37°C in a 5% CO, watersaturated air environment. At the end of the incubation period small aliquots were removed for cell counts, protein determination [ 141 using bovine serum albumin as a standard, and, in some experiments, trypan blue measurement of cell viability. The remainder of the cells were washed three times in cold phosphate-buffered saline (0.15 M NACl/O.Ol M PO,, pH 7.4), and the cell buttons were subjected to chloroform/methanol extraction, saponification, neutral lipid isolation and thin-layer chromatography for quantification of the amount of oxygenated sterol inserted [8-lo]. The neutral lipid extract was also analyzed for cholesterol content [8]. We have previously shown that repeated (five times) washing of red cells following oxygenated sterol exposure does not reduce the amount of oxygenated sterol present [9]; similar results were obtained in preliminary experiments involving lymphocytes. In addition, in-

540

cubation of lymphocytes for up to 2 h in lipoprotein-depleted medium does not diminish their cholesterol content. In certain experiments, oxygenated sterol-containing lymphocytes were subjected to a second similar incubation in various oxygenated sterol-free media, washed, and analyzed for comparison with appropriate controls to monitor the effects of lipoproteins on the egress of the compounds from lymphocytes, in a manner analogous to that previously carried out using red cells [lo]. Incubation of red cells with oxygenated sterols and their subsequent analysis were performed as previously described [8]. The standard incubation volume was 5 ml cont~ning 0.25 ml packed red cells. The latter had a cholesterol content of 333 + 18.9 ,ug (mean kS.D, n = 13). To determine the effects of oxygenated sterols on the distribution and chemical characteristics of serum lipoproteins, we added the compounds to human serum contai~ng Na,H-EDTA (0.025 g/dl). The serum was freshly isolated from blood drawn from a fasting type A donor. The final concentrations of the sterols and their ethanol solvent were 20 ,Itg/ml (50 PM) and 2% (v/v), respectively. A serum sample containing a similar amount of f4-‘4C]cholesterol was also prepared to allow comparison with a physiological sterol. After incubation at 24°C for 1 h, the sera were subjected to ultracentrifugal flotation at d 1.25 g/ml and the supernatant was then analyzed by isopycnic density gradient ultracentrifugation 1151.At the end of the run the effluent from each gradient tube was continuously monitored at 280 nm in an ISCO Model UA5 monitor [16] and 0.4-ml fractions were collected to include the main lipoprotein subclasses, VLDL (d < 1.006 g/ml), IDL (d 1.006-1.019 g/ml), LDL (d 1.019-1.063 g/ml), and HDL (d 1.063- 1.21 g/ml). The protein and cholesterol contents of each lipoprotein species were analyzed, as well as the amount of oxygenated sterol or radioactively labelled cholesterol therein ElO]. We have already described the preparation of heat-inactivated, lipoprotein-depleted serum [8]. On a preparative scale, LDL, HDL and HDL, (d 1.12- 1.2 1 g/ml) were isolated by ultracentrifugation flotation from normal human serum [ 171; each preparation was extensively dialyzed against

phosphate-buffered saline containing 0.1 mM Na,H-EDTA. They were then sterilized by passage through 0.22~pm membranes (Millipore) and stored at 4°C under N, in lift-shielded containers. To study the effects of oxygenated sterols on the exchange of red-cell membrane cholesterol with free cholesterol added to the incubation medium, the cells were incubated in lipoprotein-depleted medium as described earlier, with or without oxygenated sterol, at a final concentration of 25 PM. At the start of the incubation each flask also received free cholesterol (final concentration, 25 PM) containing 0.25 $Zi [4-*4C]cholesterol ((5-6) - IO5 cpm). Following incubation, which ranged from 1 to 20 h, the cells were washed, and the neutral lipids extracted. Aliquots of the latter were used for determination of the amounts of oxygenated sterol inserted into the red cells, for total cholesterol determination, and for measurement by scintillation counting of the amount of radioactive cholesterol from the medium which had found its way into the red cell membrane by exchange. Experiments of this kind were performed in triplicate. Results The uptake and egress of oxygenated sterols from lymphocytes

The protein and cholesterol content of the various lymph~yte lines used in our studies are shown in Table I. In general, these were not altered by incubation of the cells under the conditions we

TABLE

I

THE PROTEIN AND CHOLESTEROL VARIOUS LYMPHOCYTE CELL LINES

CONTENT

Cell type

Cells(.lOd/ mg protein)

pg Cholesterol/ mg protein

130 (normal) 968 (normal) l4588 1459 a

16.8 14.0 15.1 12.9

21.7 18.4 15.3

a Cells from patients terolemia.

with homozygous

OF

17.6

famiIia1 hypercholes-

541

TABLE

II

THE EFFECT OF TIME, CONCENTRATION LYMPHOCYTES Experiment

I.

Cell type

130

946

AND TEMPERATURE

OF OXYGENATED

Temperature

Time of incubation (min)

(“C)

20a-OHC ’ 20/320a-OHC 20~OHC 20a-OHC 20n-OHC

25 25 25 25 25

5 30 60 5 120

37 37 37 37 37

9.55 1.2 8.5rt 1.1 8.5+2.1 6.5 + 0.7 7.2+ 1.0

120 120

37 37

6.3*0.1 3.4+0.2

60 60 60

0 25 37

Concentration

946

20~OHC 20~OHC

25 12.5

III.

946

7~OHC

25

inserted inserted

STEROLS

INTO

pg oxygenated sterol inserted/ mg cell protein (mean + S.D.)

@M)

Sterol

II.

a Hy~oxycholesteroI. b By comparison with amount ’ By comparison with amount

ON THE ENTRY

(P < O.Ol)b

6.2+0.2(P<0.001)c 23.1 kO.9 (n.s.)’ 25.7+ 1.1

at 25 FM. at 37°C. n.s., not significant.

employed for the duration of the experiments (up to 2 h of incubation). The effects of oxygenated sterol concentration, time of incubation, and temperature on the uptake of oxygenated sterols by lymphocytes are shown in Table II. As in red cells [g-lo], oxygenated sterol uptake is related to the ~ncentration of the compounds in the incubation medium, and is rapid, with most of the uptake taking place within the first 5 min. Incubation of cells at 0°C decreased uptake of 7&hydroxycholesterol (and 20ar-hydroxycholesterol, data not shown) substantially by comparison with the amounts taken up by the cell at 25 or 37°C. Normal lymphocytes and lymphocytes derived from a patient with homozygous familial hypercholesterolemia did not differ in the amount of oxygenated sterol taken up when tested during the course of a single experiment with three different compounds (20a-, 7&- and 7/3-hydroxycholesterol). Our previous studies have shown that the ability of red cells to take up oxygenated sterols from lipoprotein-depleted medium is not impaired by the addition of free cholesterol to the incubation mixture, but is impeded if the lipoproteins in whole human serum are present [8,10]. We have now obtained similar results with both normal and

homozygous familial hypercholesterolemic lymphocytes (Table III). In addition, as with red cells [IO], we observed that lymphocytes which have taken up 7&hydroxycholesterol during incubation in lipoprotein-depleted medium and been washed will not lose any of the compound to the incubation medium if they are again incubated for

TABLE

III

THE EFFECT OF CHOLESTEROL ON STEROL UPTAKE

LIPOPROTEIN LYMPHOCYTE

AND FREE OXYGENATED

Lymphocytes (130) were incubated with 25 yM 7oc-hydroxycholesterol in lipoprotein-depleted medium for 90 min at 37”C, with or without various additions to the incubation mixture. Similar results were obtained using 20u-hydroxycholesterol in the place of 7a-hydroxychoIestero1, or using homozygous familial h~cholesterole~c Iymphocytes in the place of the normai cell line (dat not shown). Additions

to medium

None Cholesterol (50 pg/ml) 20% (v/v) whole serum LDL( 100 p g protein)

pg sterol inserted/mg (mean f S.D.) 13.3+ 1.7 13.1 +o 3.3rtO.l 3.4& 0.6

cell protein

542

30 min in fresh lipoprotein-depleted medium. However, 7/3-hydroxycholesterol was released into a medium containing serum lipoproteins; approximately two-thirds of the compound previously taken up by the cells emerged. Of the compounds studied, 7a-hydroxycholesterol was the one most efficiently taken up by lymphocytes in lipoprotein-depleted medium (17.4 + 5.9 pg/mg protein; n = 8) followed by 7a-hydroxycholesterol (12.3 f 1.9 pg/mg; n = 3) and 20a-hydroxycholesterol (9.7 f 3.8 pg/mg; n = 10). Of the four oxygenated sterols studied, 25-hydroxycholesterol was taken up least efficiently by lymphocytes (2.3 &-0.3 pgg/mg; n = 3). This rank order is similar to that noted using red cells (vide infra and Refs. 8, 9). Incubation of lymphocytes for l-2 h in lipoprotein-depleted medium in the presence of these oxygenated sterols did not result in loss of cell viability as measured by trypan blue dye exclusion. The effect of various lipoprotein on the uptake of oxygenated sterols by red cells and lymphocytes Having established that the conditions affecting oxygenated sterol uptake by nucleated cells in every way resemble those affecting oxygenated sterol insertion into red cells, we turned to an examination of the effects of isolated lipoprotein fractions on the entry of oxygenated sterols from

TABLE

lipoprotein-depleted medium into both cell types. When equal amounts (100 pg protein) of HDL or LDL were added to lipoprotein-depleted medium containing 25 PM 20a-hydroxycholesterol, only LDL significantly diminished entry of 20a-hydroxycholesterol into red cells when compared with the amount inserted into red cells from lipoprotein-depleted medium itself (fig 20ahydroxycholesterol inserted, mean + SD.: control, 32.7 +4.3; +LDL, 16.5 k 1.7; +HDL, 29.2 + 2.7). Larger amounts of each lipoprotein progressively impaired oxygenated sterol entry into red cells, but at each concentration LDL was 2-3-fold more effective in competing with the red cell for oxygenated sterol than was HDL (Table IV). HDL, was approximately equal to HDL in its ability to prevent oxygenated sterol uptake by red cells. Similar results were obtained using 7ol-hydroxycholesterol (Table V). LDL was also highly effective in impeding the uptake of oxygenated sterols by lymphocytes (Table III). HDL or HDL, were comparably less effective in impeding the entry of oxygenated sterols into lymphocytes, although the difference between the lipoprotein classes was less pronounced with lymphocytes that it was with red cells. The ability of lipoproteins to interfere with oxygenated sterol entry was the same with normal and homozygous familial hypercholesterolemia lymphocytes.

IV

A COMPARISON OF THE ABILITY TION OF 20wHYDROXYCHOLESTEROL

OF VARIOUS CONCENTRATIONS OF HDL INTO RED-CELL MEMBRANES

AND

LDL TO INHIBIT

THE INSER-

Two separate experiments are shown: red cells (0.25 ml) were incubated in 5 ml lipoprotein-depleted medium with 25 JLM 20whydroxycholesterol alone or in the presence of varying amounts of HDL (experiment I) or LDL (experiment II). 8 Inhibition is that compared to control. n.s., not significant. Experiment

I

II

20wHydroxycholesterol inserted (mean *SD.)

& lnhibition

( pg protein/ml)

Control (0) HDL (100) HDL (200) HDL (300) Conrol (0) LDL (50) LDL (100) LDL (200)

36.4 33 26 20.3 26.5 20.4 17.1 14.2

_

Lipoprotein

added

f 5.0 k6.0 k4.8 k 1.7 k 1.9 * 3.4 i 1.8 f 0.8

(P value)

10.7(n.s.) 28.6 (i 0.05) 44.2 ( < 0.001) _ 23 (< 0.02) 35.5 (< 0.001) 46.4 ( < 0.00 I)

543

TABLE

V

COMPARISON OF THE ABILITY OF LDL AND HDL TO INHIBIT OXYGENATED STEROL INSERTION INTO RED-CELL MEMBRANES The values given represent the mean of three experiments using each sterol and are % inhibition of oxygenated sterol insertion into red cells, by comparions with the amount of sterol entering red cells in the absence of lipoprotein. n.d. not done. Lipoprotein

20 a-Hydroxycholesterol

7P-hydroxycholesterol

pg/ml

20 50 100 200 300

LDL

HDL

LDL

HDL

12.2 25.0 41.6 46.4 n.d.

n.d. 0 14.3 28.6 44.2

n.d. 29.2 31 56 n.d.

n.d. n.d. 14.3 25.4 45.3

The isopycnic density ultracentrifugation profile of lipoproteins from a serum sample previously incubated with 20~hydroxycholesterol was the same as that from serum samples previously

TABLE

VI

THE EFFECT INCUBATION

OF VARIOUS OXYGENATED STEROLS MEDIUM WITH RED-CELL MEMBRANE

25 PM [ “C]cholesterol in red cell cholesterol Expt.

incubated with cholesterol, 25-hydroxycholesterol or 7/S-hydroxycholesterol. In addition, the protein and cholesterol distribution amongst the major lipoprotein subclasses (VLDL, IDL, LDL, HDL) as well as their cholesterol:protein mass ratios, were not significantly different in the oxygenated sterol-containing samples when compared to the cholesterol control. In contrast to the rapid uptake of oxygenated sterols by red cells and lymphocytes, with a plateau at 5 rnin of incubation and little or no change over the ensuing 2-24 h, the exchange of free cholesterol in the incubation medium with red-cell membrane cholesterol was slow and dit not completely plateau for as long as 20 h (Table VI). In general, the presence of oxygenated sterols had little effect upon red-cell membrane-medium cholesterol exchange. Of the four oxygenated sterols tested, only 7-ketocholesterol caused a slight decrease in cholesterol exchange at 1 h, while at 20 h both 20cr-hydroxycholesterol and 22-ketocholesterol caused a slight, but significant, increase in red-cell cholesterol exchange. Red-cell cholesterol content

UPON THE EXCHANGE CHOLESTEROL

Sterol (25 PM)

Time of incubation

[ I4C]Cholesterol entering red cells

(h)

(W) 4.4 8.0 13.5 28.9

None

1 2 4 20

II

None

1

5.2 + 0.2

7P-Hydroxycholesterol 20a-Hydroxycholesterol 22-Ketocholesterol

1 1 1

5.1 * 0.5 (n.s.) 6.1 f 2.1 (n.s.) 5.1 + 0.6 (n.s.)

IV

CHOLESTEROL

was added to the lipoprotein-depleted incubation medium in each experiment. There was no significant content during the course of these incubations. n.s., not significant. n.d. not done.

I

III

OF FREE

+ f ) *

0.8 0.4 1.0 1.1

None

20

28.7 f 3.7

7j3-Hydroxycholesterol ZOa-Hydroxycholesterol 22-Ketocholesterol

20 20 20

31.3 + 1.4 (n.s.) 33 f 1.2 (P i 0.05) 32.9 + 2.6 (P < 0.05)

None

1

6.8 + 0.9

pg sterol inserted/O.25 red blood cells (mean

_ _ _ _ _ 25.2 f 5.1 26.5 $ 1.4 n.d. _ 22.9 f 1.6 28.3 + 2.4 n.d. _

7-Ketocholesterol None

1 20

5.2 + 0.6 (P < 0.01) 36.2 + 2.9

n.d. -

7-Ketocholesterol

20

34.1 f 1.2 (n.s.)

n.d.

IN THE

change

ml + SD.)

544

remained constant for the duration of these experiments, as would be expected in a true exchange process. Discussion Our previous studies on the insertion of oxygenated sterols into cell membranes were carried out using human red cells [g-lo]. The present report demonstrates that the uptake of these compounds by nucleated human cells is comparable in almost every respect, and is modulated to a similar extent by such variables as concentration of oxygenated sterol in the incubation medium, temperature, time of exposure, the specific sterol studied, and the lipoprotein composition of the medium in which cellular exposure to oxygenated sterol occurs. Thus, as in red cells, uptake of oxygenated sterol lymphocytes is virtually complete within 5 min of exposure, is dependent on the concentration of the sterol in the incubation mixture, and is impeded by low temperature. In addition, the rank order of efficiency with which various oxygenated sterols enter red cells and with 7/3-hydroxylymphocytes is the same, cholesterol entering most efficiently and 25-hydroxycholesterol least efficiently. However, after exposure to various oxygenated sterols (25 PM) for 30-120 min the amount of oxygenated sterol in lymphocytes relative to their cholesterol content ranged from approximately 13% for 25-hydroxycholesterol to 90- 100% for 7P-hydroxycholesterol. By contrast, even the most efficient oxygenated sterol inserters were able to achieve a concentration in red cells equal to only about 10% of their total cholesterol content [8-IO]. With both red cells and lymphocytes the presence of free cholesterol (50 pg/ml) in the lipoprotein-depleted incubation medium does not alter the amount of oxygenated sterol which enters the cells, even though such cholesterol protects the red cell membrane against the 16% cholesterol loss which occurs after 20-24 h of incubation [8]. Substituting lipoprotein-containing serum for lipoprotein-depleted serum in the incubation mixture, however, reduces the amount of oxygenated sterol inserted into red cells and lymphocytes by 65-75%. Since we have previously demonstrated that serum lipoproteins can bind 50-80% of the oxygenated

sterols added to whole serum, we have suggested that this binding may render the sterols unavailable for entry into red cells [lo] or, in the present instance, into lymphocytes. However, the protection offered by lipoprotein against oxygenated sterol insertion into red-cell membranes is only relative, and the final equilibrium distribution of oxygenated sterol between lipoprotein and red cell membrane is such that if oxygenated sterol concentrations are increased 2.5-3-fold insertion of the compounds into red-cell membrane from lipoprotein-containing medium can equal that achieved from lipoprotein-depleted medium at lower concentrations [lo]. In the present studies, similar findings have been made with respect to oxygenated sterol entry into lymphocytes (data not shown). In addition to their ability to impede oxygenated sterol entry into cells, lipoproteins can function as oxygenated sterol acceptors and induce an equilibrium redistribution between cells and medium of oxygenated sterols previously taken up by both lymphocytes and red cells. Such a redistribution of cellular oxygenated sterol cannot be achieved by other serum proteins or bovine serum albumin [lo]. The proportion of oxygenated sterol-containing medium, all of the oxygenated sterol previously taken up by red cells or lymphocytes which can be removed from the cells by incubation in lipoprotein-containing medium (SO-65%) is similar. Since, even after 24 h of incubation in oxygenated sterol entering red cells is found in the membrane [8], the failure of some oxygenated sterol to emerge following incubation in lipoprotein-containing medium cannot be due to its sequestration in the cytosol of the erythrocyte. Whether or not the oxygenated sterol remaining in lymphocytes after exposure to lipoprotein-containing medium is also in the plasma membrane or has been translocated to the cytosol, nucleus or the membranes of intracellular organelles is not immediately apparent. Such translocation has been demonstrated for 25-hydroxycholesterol [ 181, but the degree to which it occurs may differ from one oxygenated sterol to another, and may in part be related to the amphipathic properties of the various compounds. While the effects of oxygenated sterols on redcell membrane morphology and surface area are

545

clearly independent of the metabolic effects which the compounds have upon the cholesterol biosynthetic pathway [6], the same cannot be said for the changes in membrane function which oxygenated sterols impose on nucleated cells. Since the ability of oxygenated sterols to inhibit mouse L-cell endocytosis and T-lymphocyte cytotoxicity is easily reversible by mevalonic acid [2,4], these effects are probably a direct result of the ability of the sterols to inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase, the enzyme which catalyzes cellular mevalonic acid synthesis from its immediate precursor. Other effects of oxygenated sterols on nucleated cells, such as their cytotoxic effects [5,6], their ability to inhibit granulocyte chemotaxis [7] and their ability to inhibit the formation of E rosettes by T lymphocytes [6], are unaffected by mevalonic acid and for this, and other other reasons [5-71, appear to be due to mechanisms independent of the ability of oxygenated sterols to interfere with mevalonic acid and sterol biosynthesis. We have suggested that these latter inhibitory effects of oxygenated sterols on nucleated cell membrane functions are a consequence of the presence of the sterols in the cell plasma membrane [5-71. Our present studies suggest, however, that while the presence of oxygenated sterols in nucleated cells may be a necessary component of the ability of oxygenated sterols to produce these latter alterations in cell membrane function, it is not by itself sufficient. Uptake of oxygenated sterols by nucleated cells is virtually compete within 5 min, while the alterations in membrane function develop over periods of time ranging from 15 min to 18 h [5-71. Thus, in addition to entry of oxygenated sterols into cells, some other mechanism such as translocation of the compounds to some functionally important intracellular site, the secondary induction of a membrane change made possible by their presence [6,19], or their metabolic conversion within the cell to an active product, must be involved. While homozygous familial hypercholesterolemia cells are deficient in LDL receptors, and are resistant to the modulating effects of LDL cholesterol on hydroxymethylglutaryl-CoA reductase activity and sterol synthesis, they are as sensitive as normal cells to the metabolic effects of

oxygenated sterol on cholesterol metabolism [12,20]. In addition, as our results demonstrate, oxygenated sterol uptake by these cells is in every way comparable to the uptake of the sterols by their normal cell counterparts. Clearly, as this and other aspects of our studies indicate, uptake of oxygenated sterols by nucleated or non-nucleated cells is independent of the LDL receptor mechanism. Similar conclusions have been reached in a study of the mechanism of entry of benzo[ alpyrene into cultured mammalian cells [21]. Our studies employing purified LDL and HDL have confirmed our earlier speculation that the decreased ability of oxygenated sterols to enter red-cell membranes from lipoprotein-containing as opposed to lipoprotein-depleted medium is due to the competitive binding of the sterols which lipoproteins exert [lo]. Since both LDL and HDL have been shown to bind oxygenated sterols [lo], it is not surprising to discover that each of them, in its isolated form, can effectively impede oxygenated sterol entry into red cells and lymphocytes. Our studies reveal that, on the basis of protein stoichiometry, LDL is approximately 2.5-4 times as effective as HDL in preventing oxygenated sterol entry into cells, regardless of the sterol being studied. When compared on the basis of their lipid content or their proportion normally present in serum [22], however, the difference in their ability to bind oxygenated sterol or to prevent their entry into cells is less striking. The hydrodynamic properties of the lipoproteins particles, as well as their sterol and protein composition, are not grossly disturbed by the presence within them of oxygenated sterols under the conditions we have employed. The kinetics of cholesterol exchange between the medium and the red-cell membrane, when 10 pg/ml free cholesterol is present in lipoprotein-depleted medium and the red cell cholesterol content remains constant for the duration of the incubation, resemble those described by others [23], and contrast sharply with the kinetics of oxygenated sterol insertion into red cells. Thus, while the latter is abrupt and is virtually complete within 5 min, the former continues at a gradually decelerating pace over a period of 20 h. In contrast to the observations of Bing and co-workers [24,25], who, under different conditions, noted that 7-keto-

546

cholesterol inhibited cholesterol exchange between plasma and arterial endothelium, we found little or no alteration in red-cell cholesterol exchange by any of the oxygenated sterols tested. Since oxygenated sterols possess angiotoxic properties [6], are suspected of being atherogenic [26], and are probably a component of the human diet [27], as well as being, in some instances, normal products of sterol metabolism [1,28], the modulating effects of serum lipoproteins on insertion of oxygenated sterols into cells may have physiological significance. The ability of lipoproteins to prevent oxygenated sterol entry into cell membranes, and the consequent oxygenated sterol-induced derangements of cell membrane structure and function, may be of importance in preventing pathological changes inducible by oxygenated sterols in vivo. Acknowledgements This work was supported by The Council for Tobacco Research (Grant No. 1363), the USPHS (P30 AM-26678-03 and HL 18577-07) and the Nalco Cancer Research Fund. References 1 Kandutsch, 2 3 4 5 6

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