ARCHIVES Vol.

OF BIOCHEMISTRY 189,

No.

1, July,

AND pp.

51-62,

Fluorescence-Polarization

DONALD

Departments University

BIOIBHYSICS 1978

Measurements on Normal and Mutant Human Skin Fibroblasts

F. HAGGERTY, VIJAY REYNOLDS, AND of Psychiatry of California,

K. KALRA, GEORGE POPJAK, FRANCESCO CHIAPPELLI

and Biological Chemistry, School of Medicine Los Angeles, California 90024, and Department Southern California, Los Angeles, California Received

October

13, 1977; revised

December

and Molecular of Biochemistry, 90033

ELWOOD

E.

Biology Institute, University of

12, 1977

Measurements of fluorescence polarization in intact diploid skin fibroblasts after exposure to l,&dipbenyl-1,3,5-hexatriene were used to estimate the fluidity of the lipid phase(s) of cellular membranes. The membrane lipids of cells derived from four patients with homozygous familial hypercholesterolemia were in a more fluid state than those of cells obtained from 13 other individuals of normal and nonrelated mutant genotypes when all cultures were grown on medium with native serum. The only other cell type having membrane lipids of increased fluidity under these conditions was one fibroblast line derived from a patient with the Lesch-Nyhan syndrome. Examination of two additional nonconsanguinous lines of Lesch-Nyhan tibroblasts, however, revealed that an abnormally high level of lipid fluidity was not a common property of the membranes of cells of this genotype. Incubation of cultures in medium containing lipid-depleted serum (virtually devoid of lipoprotein-bound sterol) caused a reversible increase in the fluidity of the membranes of normal cells to values similar to those of the hypercholesterolemic cells, but had no effect on the membranelipid fluidity of the latter. By contrast, exposure of cultures to cholesterol not bound to lipoprotein in serum-free medium resulted in a decrease in the lipid fluidity of the membranes of both normoand hypercholesterolemic fibroblasts.

mixed population” of leukocytes of heterozygous FH patients responded to incubation for 6 to 18 h in lipid-depleted serum with an abnormally high induction of HMG-CoA reductase (17, 18) and that this effect was preceded by a loss of newly synthesized cellular cholesterol greater than that observed with similarly treated leukocytes of normal individuals (19). These observations clearly indicate an abnormality of plasma and/or intracellular membranes in FH: an inability to bind LDL and hence transfer LDL-cholesterol into the cell and an impairment of the ability of the cells to adequately retain endogenously synthesized cholesterol. It is known that the cholesterol content of cultured fibroblasts of homozygous familial hypercholesterolemics is lower than normal even though they may

Studies of cultured human fibroblasts from normal individuals and individuals homozygous for familial hypercholesterolemia (FH)’ by Brown et al. revealed that while the normal cells possessedhigh-affinity surface receptors for low-density lipoproteins (LDL), FH cells were devoid of these receptors [(l-5); for reviews, see (6-a)]. Moreover, LDL, taken up and degraded by the normal cells (2, 3, g-11)) suppressed the induction of 3-hydroxy-3methylglutaryl-CoA (HMG-CoA) reductase, but failed to do so in FH (2, 3, 10-15) fibroblasts. Fogelman et al. found that a ’ Abbreviations used: FH, familial hypercholesterolemia; HMG-CoA, 3-hydroxy-3-methylglutarylcoenzyme A, MEM, Eagle’s minimum essential medium; DMEM, Dulbecco’s modified MEM; PBS, phosphate-buffered saline; PL, phospholipid; TG, triglyceride; CE, cholesterol ester; GSL, glycosphingolipid; LDL, low-density lipoprotein; sucrose-TEA, 0.25 M sucrose-l mM triethanolamine-HCl (pH 7.4).

’ It was shown (16) that the most active cells with respect to cholesterol synthesis and inducibility of HMG-CoA reductase were the monocytes. 51 0003-9861/78/1891-0051$02.00/O Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

52

HAGGERTY

be synthesizing cholesterol at rates 20 to 50 times higher than normal (20, 21). Leukocytes of heterozygotes, when freshly isolated, contain no more cholesterol than do the cells of normal individuals (19, 22), but they lose more sterol than normal cells in a medium containing lipid-depleted serum (1%. We were prompted by the above briefly summarized observations to examine the lipid fluidity of membranes of cultured fibroblasts from various sources, including FH, by measurements of fluorescence polarization after treating cells with the fluorophore 1,6-diphenyl-1,3,5-hexatriene. MATERIALS

AND

METHODS

ET

AL.

4L

B

21

:pJ.?.& I 31

Cell cuhre. A line of skin fibroblasts (line 5) originating from a patient with the receptor-negative form of homozygous FH (P.A., a patient of Dr. N. B. Myant of the Lipid Research Unit of the Medical Research Council at the Royal Postgraduate Medical School, London, England) (24) was generously provided by Dr. Daniel Steinberg of the University of California at San Diego. Three additional lines of this genotype (lines 26, 27, and 28) were obtained from the Human Genetic Mutant Cell Repository (Camden, New Jersey) through the cooperation of Dr. Joseph L. Goldstein of the University of Texas at Dallas. These cell lines were designated GM-701, GM-1915, and GM2000, respectively; they are noted as lines 26-28 here. Lines 8, 12, 13, and 22 were derived in this laboratory from explants of foreskin&obtained from the Neonatal Nursery at the UCLA Medical Center, of presumed normal infants. Although, for all of the experiments, the cells of lines 5, 12, 13, 24, and 25 (except for line 5 in the experiment of Fig. 1A) were used before the 20th passage (45 to 60 population doublings), control experiments indicated that the number of passages had no effect on fluorescence-polarization parameters. Lines 7,14 through 20,21, and 24 and 25, respectively, were obtained from Drs. John Blass, Stephen Cederbaum, Barbara Crandall, and Hayato Kihara of this institution; these lines were derived either from normal individuals or from patients with heritable metabolic disorders other than FH (cf. Table II). Lines 7, 8, and 21 were free of Mycoplasma, as judged by standard techniques of microbial cultivation and by measurements of cellular uracil uptake and nucleoside phosphorylase activity (25); these same methods, however, revealed that line 13 was infected. That the presence or absence of Mycoplasma did not affect the fluorescence-polarization values obtained with the cultures is apparent from the data in Table II. Stocks of all cell lines were grown in surface culture in 75-cm’ flasks (Falcon or Lux) on Dulbecco’s moditied Eagle’s minimum essential medium (DMEM)

3.2

[TEMPERATURE

33

3.4

3.5

L 3.6

(“K)]-‘(x103)

FIG. 1. Arrhenius plots ofp values calculated from measurements of fluorescence polarization on fibroblasts of normoand hypercholesterolemic genotype. Cultures of fibroblasts were grown in duplicate in MEM (A) or in triplicate in DMEM (B and C) medium supplemented with 10% fetal bovine serum and were harvested in parallel for the measurement of fluorescence polarization as described under Materials and Methods. For each line, light scattering was estimated on the cells from one flask, while fluorescence polarization was measured in duplicate on the cells from each remaining flask at the temperatures indicated on the abscissa. The means of the resulting p values (X 10) are plotted on a log scale vs the reciprocal of absolute temperature. Where the locus of points for the data from line 5 appears to have two slopes (A and B), the straight line through each segment was calculated separately. The coefficient of determination, 3, for the points comprising each line or segment is also shown. The genotypes of the lines used are the following: line 5 (O), homozygous FH; line 7 (x), pyruvate dehydrogenase deficiency; lines 8 (A) and 12 (A), normal Caucasian; and line 13 (V), normal Negro.

(Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco). On occasion, cells were maintained on modified Swim’s medium S-77 (26, 27) containing 5% fetal bovine and 10% horse sera, but the intermittent use of this medium did not affect fluorescence-polarization parameters in subsequent measurements. Cells were passaged for experiments into 75-cm’ flasks containing 10 to 20 ml of medium, as needed, after a 5min exposure of confluent stock cultures to a solution of 0.05% (w/v) trypsin, 0.02% (w/v) sodium EDTA in citrate-saline (26) at 37°C. Media were renewed or changed every 2 to 3 days. Although cultures were usually harvested at confluency for measurements of

MEMBRANE-LIPID

FLUIDITY

fluorescence polarization, control experiments revealed that the groswth status of the cells had no effect on the parameters obtained. (For example, in the experiment of Fig. IC, the cultures of line 12 were subconfluent, whereas those of line 13 were heavily postconfluent.) Cultures to be exposed to serum-free DMEM medium supplemented with cholesterol were first preincubated for 21 to :23 h in 15 ml per flask of the serumfree medium alone. Under these conditions, the cultures remained viable, but cellular replication ceased. By the end of the preincubation period, the cultures of line 5 were postconfluent, while those of line 12 were not yet confluent. Twenty-one to twenty-four hours before harvesting for fluorescence-polarization measurements, all cultures were given a renewal of serum-free medium, and an ethanolic solution of recrystallized cholesterol was added to the appropriate flasks to a final concentration of either 50 or 100 pg of sterol/ml and 1.5% (v/v) of ethanol in the medium. At these concentrations, the sterol did not remain in solution, but formed a somewhat turbid suspension. Neither the cholesterol nor the ethanol affected cellular morpholog,y or attachment of the cells to the substratum, and ethanol (1.5% v/v) did not diminish cellular growth rates in serum-containing medium. The turbidity in cell suspensions that resulted from the use of concentrations of sterol higher than 100 ag/ml caused a degree of light scattering that prevented reproducible measurements of fluorescence polarization. Human full and lipid-depleted AB-negative serum, obtained and prepared as described previously (17), were the generous gift of Dr. Alan Fogelman of the UCLA Department of Medicine. Cholesterol was purchased from Pfaltz-Bauer and was recrystallized twice from ethanol. Fluorescence-polarization measurements and calculation ofp values. 1,6-Diphenyl-1,3,5-hexatriene has been used as a fluorescent probe to measure the fluidity of lipid bilayers in both liposomes and biological membranes (28, 29). Surface cultures of fibroblasts were washed twice with phosphate-buffered saline (PBS), pH 7.2, and incubated with 2 PM diphenylhexatriene (added in tetrahydrofuran) for 60 min at room temperature, as previously described for mouse 3T3 cells (30). The cells were then washed with PBS and detached from the substratum by a 5-min exposure to 0.02% (w/v) EDTA in PBS. After collection and washing by centrifugation, the cells were resuspended in PBS. Fluorescence polarization and intensity were measuredwithanh4PF-4Perkin-Elmerspectrofluorometer equipped with polarizers and a thermostatically controlled cuvette holder. The temperature within the cuvettes was recorded with a thermistor probe. The excitation wavelength was 360 nm and the emission was measured at 426 nm. The excitation and emission slit widths were set for 5 nm. The excitation period

OF

53

FIBROBLASTS

was 10 s, in order to minimize reversible photoisomerization (29). Samples were stirred with a plastic rod before polarization values were measured to ensure a homogeneous dispersion of cells. The fluorescence-polarization value p was calculated from the equation (31):



1v.v - zv.h &/~h.h) zv,v + 1v.h (&.v/&,.h)’

=

where Z is the corrected fluorescence and the subscripts v and h indicate values obtained with a vertical or horizontal orientation, respectively, of the excitation and analyzer polarizers, in that order. The corrected fluorescence was determined by subtracting the intensity of light measured with unlabeled control cells from the intensity observed with the labeled cells. In this manner, error in the fluorescence-polarization measurements resulting from scattering of the polarized excited light is minimized. The maximum error of such corrected values is usually less than &5%. Since, in accordance with the Perrin equation (Table I), p is directly proportional to microviscosity (11) (inversely proportional to fluidity), the results of most experiments have been expressed in terms of p, Table I, however, gives the values for 7 corresponding to the p TABLE

I

FIJJOKESCENCE-POI,ARIZATION VAIAJES AND MEMBRANE-LIPID MICROVISCOSITIES OF SKIN FIRHORLASTS FROM A NORMAI. INDIVIDIIAI, AND A PATIENT WITH HOMOZYGO~S FAMIIJAI, HYPERCHOLESTEROI.EMIA” Line

12 5

Genotype

Normal Homozygous familial hypercholesterolemia

Polarization (P)

Anisotropy (r)

Microviscosity (4 WV

0.243 0.197

0.176 0.140

2.47 1.62

“ After continuous growth in medium with full serum, cultures from lines 5 and 12 were labeled and harvested, and 4 X 10’; cells from each were prepared for fluorescence-polarization measurements at 25°C as described under Materials and Methods. The 7 value for the probe was determined from the relative fluorescence intensities at different temperatures with an r” value of 11.4. Microviscosity was calculated from the Perrin equation:

o/r = CWTT/~, where r and rD are the measured and limiting fluorescence anisotropies, respectively; T is the absolute temperature; 7 is the excited-state lifetime; n is the microviscosity; and C(r), a function of r, is a shape parameter for the fluorophore, here being equal to 8.6 x lo” P.deg-‘.s-‘.

54

HAGGEHTY

values obtained with two representative cell lines among those examined. Preparation of plasma-membrane-enriched fractions and measurement of fluorescence polarization. Since an earlier study showed that the fluorescent probe used in these experiments could be taken up by an intact cell and that, under certain conditions, the fluidity of internal cellular lipids could contribute significantly to overall measured fluorescence-polarization values (32), we prepared subcellular fractions enriched in plasma membranes in order to investigate the degree to which the fluorescence polarization of intact cells reflected the lipid fluidity of cellular membranes in general and of the plasma membrane in particular. Plasma membranes were isolated by a method similar to that described for muscle fibroblasts by Schimmel et al. (33). Surface cultures of the fibroblasts were washed twice with 10 ml of PBS and the cells from each flask were scraped with a rubberjacketed spatula into 5 ml of PBS, transferred to a 50 ml centrifuge tube with two IO-ml washings with buffer per group, and collected by centrifugation at 295g for 10 min. The cell pellet (0.4 g) was suspended in 5 ml of 0.25 M sucrose, 1 mM triethanolamine-HCI (pH 7.4) (sucrose-TEA) and homogenized in a Dounce homogenizer with the tight B pestle (Kontes Glass Co., Vineland, New Jersey). Twenty excursions were sufficient for 80 to 90% breakage, as judged by phasecontrast microscopy. The homogenate was sedimented by centrifugation at 1800~ for 10 min and the supernat,ant solution was removed; the pelleted material was again homogenized and the centrifugation repeated. The combined supernatants were then centrifuged at 30,OOOg for 45 min. and the resulting pellet was suspended in 0.5 ml of sucrose-TEA and layered over a discontinuous sucrose gradient of the following composition: 0.5 ml of 55% sucrose and 0.9 ml each of 40, 32, 27, and 20% sucrose. The centrifugation was carried out in an SW 50.1 rotor at 44,000 rpm (200,OOOg) for 90 min. Bands appearing at the 20 to 27 and the 27 to 32% interfaces were removed with a Pasteur pipet, diluted to 8 ml with sucrose-TEA, and then centrifuged at 144,OOOg for 60 min. The resulting pellet was suspended in sucrose-TEA and assayed for ouabain-sensitive Na’-, K’-ATPase activity. ATPase activity was measured in a reaction mixture containing the sample solution (40 to 60 ag of protein), 10 mM Tris-HCI (pH 7.4), 150 mM NaCl, 15 mM KCI, 5 tnM Mg&, 10 mM ATP, and water, in a final volume of 300 nl. For the estimation of ouabain inhibition, the samples were preincubated with 1 mM ouabain for 10 min at 37°C before the addition of ATP. The reaction was terminated by the addition of 0.5 ml of 10% (w/v) aqueous trichloroacetic acid. The released orthophosphate was estimated by the method of Fiske and SubbaRow (34). One unit of enzymatic activity is defined as the release of I nmol of orthophosphate per minute. In addition to assaying ouabain-sensitive Nat-, K’-ATPase in whole homogenates and plasma

ET

AI,

membrane-enriched fractions, succinic dehydrogenase and glucose 6-phosphatase were measured as markers for contamination of the latter preparations by mitochondria and endoplasmic reticulum, respectively. Succinic dehydrogenase was assayed by the method of King (35), and glucose 6-phosphatase according to H ubscher and West (36). Measurement of cholesterol content of cells. Triplicate cultures of each line to be analyzed were grown to postconfluency in 75.cm’ flasks in medium supplemented with full serum. After a fivefold washing with 10 ml of Tris-NaCl-albumin buffer (50 mM Tris-Cl, 0.15 N NaCI, 2 mg/ml of bovine serum albumin, pH 7.4) and a final washing with the same buffer without albumin, the cells from each flask were detached by scraping with a rubber-jacketed spatula into 3 ml of albumin-free buffer, transferred to a 15-ml graduatedglass Teflon-capped centrifuge tube with two 5-ml washings with buffer, sedimented by centrifugation for 10 min at 147Og, and stored at -1O’C. After subsequent thawing, the cell pellet was dissolved in 500 ~1 of 0.1 N KOH at 37°C for 16 h, and 25 aI were removed in duplicate for the determination of total cellular protein content. To the remaining 450 ~1 was added 300 ,a1 of 8.18 N KOH plus 1.4 ml of absolute methanol, to give final concentrations of 1.2 N and 65%. respectively; then the sample was heated for 3 h to saponify total cellular lipids, After the addition of 80 nl of a methanolic solution containing 40 ng of coprostanol (Applied Science Laboratories) to provide an external mass standard, the nonsaponifiable lipids were extracted three times with 4 vol of petroleum ether; the upper, organic phases were combined, reduced in volume under nitrogen at 40°C and transferred to a tared vial; and the final lipid residue was redissolved in 206 al of chloroform. One microliter of this solution was injected into a Hewlett-Packard Model 5830A gas-liquid chromatograph, equipped with a hydrogenflame detector and an automatic peak integrator, containing a 6-ft glass column packed with 3% OV-17 on 100/120-mesh Gas-Chrom Q (Applied Science Laboratories), and operating isothermally at a column temperature of 260°C and a gas flow of 33 ml of helium/min. Under these conditions, the retention times of coprostanol and cholesterol were 12.47 f 0.08 and 14.40 + 0.12 min (mean k SD), respectively, and the total amount of cholesterol in the sample was calculated from the ratio of the areas under the two chromatographic peaks. The cholesterol content of the cells was expressed as micrograms of sterol per milligram of cellular protein. Protein determinations. The protein concentrat.ions of solubilized extracts were estimated either by the technique of Lowry et al. (37) or by a modification of a more recently published procedure employing Coomassie brilliant blue G (38). With the latter method, a constant volume of the reagent solution [Coomassie brilliant blue G-250, 0.01% (w/v); ethanol, 4.7% (v/v); H:gPO,, 8.5% (v/v)], usually 1 or 3 ml, was

MEMBRANE-LIPID

FLlJII>ITY

mixed with 0.1 ml of an appropriate dilution of the unknown sample in Tris-KC1 buffer (26), and absorbance at 595 nm was read immediately in a Unicam Model SP-1800 spectrophotometer, along with that of standard samples containing known amounts of bovine serum albumin (Sigma; fatty acid free, No. A-6003) in the same volume of buffer. With each assay, a standard curve of log absorbance vs log albumin content was constructed according to the method of least squares, from which the protein concentrations of unknown samples whose absorbance fell within the linear portion of the curve were read. Statistical eualuations. The straight lines providing the best fit to each of the sets of data shown in Fig. 1 according 1;o the method of least squares, along with the corresponding coefficients of determination (3) and the SD, SEM, and t values used for the Student t test in ‘Tables II, III, and IV, were calculated by the use of the appropriate programs with a Hewlett-Packard HF’-25 programmable pocket computer w-u. RESULTS

Examination of membrane-lipid fluidity of cells of various genotypes after continuous cultivation in medium containing fetal bovine serum. In initial experiments, fluorescence-polarization measr. rements on fibroblasts from a patient with homozygous FH were compared with those obtained from four control lines: three derived from presumed normal foreskin (two Caucasian, one Negro) and one originating from a patient with deficient pyruvate dehydrogenase activity and high blood-lactate levels. Fluorescence-polarization measurements were taken at different temperatures on each sample, and Arrhenius plots of the log of the p values vs the reciprocal of the absolute temperature were made. Figure 1 shows the results obtained in three separate experiments,. Whereas the values for logp decreased linearly with decreases in the reciprocal absolute temperature for all three cell lines in each experiment, the logp values at a given temperature were always higher for either of the two control cells than for the mutant cells, thus indicating that the membrane lipids of the FH cells had a lower microviscosity, or were in a state of greater fluidity, than those of the normal cells (Table I). This difference was not affected either by the passage number of the lines or by the culture density of the cells during a given passage. Although the

OF

FIBKOBLASTS

55

discontinuity in the slope of the curves for line 5 in Figs. 1A and B (see legend) suggests the occurrence of a phase transition in the membrane lipids of these cells at about 30°C this break was not seen in all experiments (cf. Fig. 1C). The data in Table II show that the fluorescence-polarization values obtained on subcellular fractions enriched in plasma membranes were not significantly different from the values obtained on intact fibroblasts, suggesting that the measurements made in whole cells reflected mainly the properties of lipids of plasma membranes rather than of intracellular membrane structures.” In addition to the ouabain-sensitive Na+-, K’-ATPase as a measure of enrichment of plasma membranes (cf. Table II), we also assayed succinic dehydrogenase and glucose 6-phosphatase as markers for contamination of plasma membranes by mitochondria and endoplasmic reticulum, respectively. The maximum contamination of plasma membranes from normal and FH cells with mitochondria, as judged by a comparison of the succinic dehydrogenase activity of whole homogenates with that of plasma membranes, was 10.4% (mean +SD, 9.3 + 1.4%). In two plasma membrane preparations from normal cell lines (lines 8 and 13) glucose 6-phosphatase could not be detected. In the preparation from the homozygous FH cells (line 26), glucose 6phosphatase activity was 12.9% that of the unfractionated homogenate. Thus, we are confident that our measurements reflect properties of the hydrophobic regions of plasma membranes rather than of any other cellular structure. Van Blitterswijk et al. (40) showed recently that the microviscosities, measured by DPH fluorescence, of ,’ The Na’-, K’-ATPase activities of plasma membrane-enriched subcellular fractions prepared by the method of Schimmel et al. (33) were labile to overnight storage at -20°C. Thus, the membranes used in the experiment of Table II were assayed on the day of their preparation. In contrast, the ATPase activities of membrane fractions obtained by the zinc ion procedure of Warren (46) remained stable in the frozen state for up to 3 days. Moreover, the fluorescencepolarization values and ATPase activities of membranes freshly prepared by either technique were respectively equivalent.

56

HAGGERTY TABLE

COMPARISON Line

AL

ET II

OF FI.~JORF,SCENCE-POI.AHIZATION VALIJES OBTAINED WITH INTAC’I’ FIRHORLASTS THEIR SIJRCEJLULAR FRACTIONS ENRICHHI IN PLASMA MEMBRANES” Genotype

Ouabain-sensitive Na’-. K+ATPase specific activity (units/mg of protein)

22

Normal, Sian

Cauca-

13 28

Normal, Negro Homozygous FH

Intact cells

Plasma membranes

N.D.h

N.D.

32.8 32.8

191 148

p Value

Intact

(X 10:‘) f SD

cells

Plasma membranes

Experiment I 242 f 3 (2)

245 + 13 (2)

Experiment 2 238 + 5 (4) 206 k 6 (4)

259 f 11 (4) 208 -+ 11 (4)

n For each experiment, 8 to 10 replicate 75-cm” cultures of the indicated postconfluency in medium with full serum. Cultures were harvested; plasma fractions were prepared; and fluorescence-polarization values, Naf-, K’-ATPase were measured as described in the text. ’ Not done. ” Intact cells vs plasma membranes, same cell line. ’ Intact cells, line 13 vs line 28. e Plasma membranes, line 13 vs line 28.

liposomes prepared from the total lipids of plasma membranes of thymus-derived ascitic leukemia (GRSL) cells and of thymocytes “were the same as, or sometimes only slightly lower . . . than those of the corresponding intact membranes”. Van Blitterswijk et al. (40) concluded that there was “virtually no influence of (glyco)proteins on the lipid fluidity.” Table III summarizes the results from experiments designed to assessthe statistical significance of the observed differences in p values obtained with FH and control cells (Experiment 1). In this experiment, fibroblasts from the patient with FH were grown in parallel with cells from 11 control lines, the 3 lines from normal foreskin examined previously plus 8 other lines derived from normal individuals and from patients with heritable disorders other than FH (cf. the genotypes listed in Table III). The p values obtained from fluorescence-polarization measurements at 26’C on cells from 10 of these lines did not differ from each other (p = 0.243 f 0.012; mean f SD), but were significantly higher than the p values

Statistical

ANI) WITH

evaluation dent t test)

(Stu-

t

df

P

3.44 0.201 8.42 6.54

6 6 6 6

O.Ol’ m.5 0.01). To investigate further the possibility that lower than normal p values might be a property of all Lesch-Nyhan fibroblasts, two additional lines derived from nonconsanguinous individuals of this genotype (lines 24 and 25) were obtained and grown in parallel with the cells of lines 5, 12, and 21. As is clear from the fluorescence-polarization measurements in Experiment 3 of

TABLE

III

STATISTICAL COMPARISON OFJJ VAISIES ORTAINED FROM MEASUKEMENTS OF FIAIOHESCENCE POLARIZATION ON CELLS OF VARIOUS GENOTYPES” Line

p Value

Genotype Experiment

5

Homozygous

(X 19%) k SE

1

FH

197 (2) f SE, 197 +- 0.33 (2) 262 (1) 256 (1) 247 (1) 248 (1) 253 (1) 232 (1) 238 (1) 230 (1) 230 (1) 237 (1) Mean f SE, 243 f 3.6 (10) = 10, te = 12.2, P’ < 0.001)’ 153 (1) Mean

a* 12 13 14 15 16’ 17 18’ 19’ 20

Normal, Caucasian Normal, Caucasian Normal, Negro Normal Heterozygous citrullinemia Homozygous homocystinuria Heterozygous citrullinemia Heterozygous citrullinemia Normal Uncharacterized lactic acidosis

21

Lesch-Nyhan

5

Homozygous

(cp syndrome Experiment

2

FH

13

Normal,

21

Lesch-Nyhan

5 12 21 24 25

Homozygous FH Normal, Caucasian Lesch-Nyhan syndrome Lesch-Nyhan syndrome Lesch-Nyhan Syndrome

13 21

Normal, Negro Lesch-Nyhan syndrome

26

Homozygous

209 k 5.8 (2) (df = 4, t = 7.36, 0.01 > P > 0.001)’ 253 f 4.2 (4) (df = 5, t = 15.5, P < 0.001)~ 161 f 4.1 (3) (df = 3, t = 5.38, 0.02 > P 1 0.01)’

Negro syndrome Experiment

3’ 196 242 195 239 239

Experiment

4k

(df = 4, t = FH (df = 3, t = 27

Homozygous

FH

28

Homozygous

FH

(I) (1) (2) (2) (2)

(df=

4, t =

(df=

4, t =

237 191 6.32, 0.01 > P 196 11.0, 0.01 > P 198 7.11, 0.01 > P 199 6.67, 0.01 > P

+ 3.7 (3) + 6.4 (3)

> 0.001)' + 1.0 (2) > 0.001)’ f 4.2 (3) > 0.001)’ +- 4.5 (3) > 0.001)’

n Replicate cultures of each line of tibroblasts were plated in DMEM medium supplemented with 10% fetal bovine serum, grown continuously on that same medium, and harvested in parallel for measurement of fluorescence polarization at 26°C. Unless indicated otherwise, the cultures were confluent at the time of harvesting, and each p value represents the mean of triplicate determinations made on the number of cultures shown in parentheses, with a separate culture being used to estimate light scattering. For Experiment 2, the statistical comparison was made among all three sets of data; for Experiment 4, the data from line 13 were compared with those from each of the other lines. ’ Subconfluent. ’ Heavily postconfluent. “Degrees of freedom = (nl - 1) + (n2 - 1). ’ From the Student t test. ‘Line 5 vs line 8 plus lines 12 through 20. p Line 5 vs line 13. ’ Line 13 vs line 21. ’ Line 5 vs line 21. ’ Single measurement per culture. ’ Duplicate measurements on two cultures; triplicate measurement on third culture when present. ’ Versus line 13. 57

58

HAGGER’I’Y

Table III, the lowp values associated with the cells of line 21 were atypical of Lesch-Nyhan fibroblasts, since normal values were found with the cells of lines 24 and 25. Because we found one cell line (line 21), among all the control lines examined, that exhibited anomalous p values comparable to those of the FH cells, the possibility that line 21 represented an example of the FH genotype, in addition to the Lesch-Nyhan anomaly, could not be ignored. We thus attempted to resolve this question using three separate approaches: (i) We obtained, from the Human Genetic Mutant Cell Repository of NIH, three additional lines of FH cells from unrelated individuals (lines 26, 27, and 28) and examined their membrane-lipid fluidities by fluorescence polarization; (ii) we determined the total cellular cholesterol content of lines 13, 21, and 26 after growth of the cultures under conditions producing differences in their p values (i.e., in medium with full serum); and (iii) we investigated the effects on membranelipid fluidity of conditions known from other studies to modulate cellular cholesterol content (see the following section). The fluorescence-polarization data obtained with cultures from lines 26, 27, and 28 in Experiment 4 of Table III conclusively demonstrate that abnormally high membrane-lipid fluidities (i.e., low p values) are a general characteristic of skin fibroblasts from patients with the receptor-negative form of homozygous FH: Thep values measured with the three new FH lines were comparable to those obtained with line 21 in this experiment and were significantly lower than those of line 13 (the control line) (P < 0.001 for all three lines). Since two earlier reports from other laboratories indicated that fibroblasts from FH individuals contained lower levels of cholesterol per milligram of cellular protein than did cells from normal subjects when cultures were grown in medium containing full serum (20, 21), we determined the cholesterol contents of the cells of lines 13, 21, and 26 under this same condition. Whereas, in confirmation of the earlier findings (20, al), the concentration of total (i.e., esterified and nonesterified) cholesterol was sig-

ET

Al,

nificantly lower in the FH cells (line 26) than in the normal fibroblasts (line 13) (21.2 f 0.2 and 27.6 + 0.5 pg/ml of protein, respectively; t = 18.8, df = 4, P < O.OOl), the cholesterol content of the anomalous Lesch-Nyhan cells (line 21) was as great as, or greater than, that of the latter (30.9 + 3.8; t = 1.47, df = 4, 0.3 > P > 0.2). Consequently, it would appear unlikely that the cells of line 21 harbor a defect in cholesterol metabolism similar to that of the FH fibroblasts in addition to bearing the Lesch-Nyhan mutation. Thus we conclude that while an increased fluidity of membrane lipids may be related to an abnormality of cholesterol metabolism (and a low cellular cholesterol content) in FH fibro-

3oor

150 1.1

i- Sth’

1--012345

-

I

i

~

DAY

2. Effect of the presence or absence of lipoprotein-bound cholesterol on p values obtained with control and homozygous fibroblasts. Replicate cultures of cells from lines 5 (homozygotes = circles) and 13 (controls = triangles) were grown in DMEM supplemented with 10% full human AB-negative serum for 5 days, when (Day 0) some of the cultures of each line were transferred to DMEM with lipid-depleted human serum (open and half-filled symbols), while others were kept on the medium with full serum throughout the entire experiment (closed symbols). On Day 3, some of the former cultures were returned to the medium with full serum (half-Nled symbols), while the rest of were kept on medium with lipiddepleted serum (open symbols). On Days 0, 3 and 5, fluorescence-polarization measurements at 25°C were made on three replicate cultures for each experimental condition. FIG.

MEMBRANE-LIl’II>

FLUIDITY

blasts, such a correlation is not universal for all genotypes. Reversible changes in overall fluidity of cellular mem#brane lipids accompanying growth in medium depleted in lipoproteinbound sterol. In view of the significant and reproducible differences in cellular membrane-lipid fluidity between fibroblasts of presumed normocholesterolemic and FH individuals grown continuously in medium containing native fetal bovine serum, we examined the effect of deprivation of extracellular lipoprotein-bound sterol on this parameter in both cell types. The media used in these experiments consisted of the standard basal medium (DMEM) supplemented with either full or lipid-depleted AB-negative human serum (lo%, v/v). In the experiment of Fig. 2, control and FH fibroblasts were plated directly into medium with full serum and grown to near confluency (Day 0). Some of the cultures were then given medium with lipid-depleted serum, while others were kept on medium with full serum throughout the entire experiment. Finally, on Day 3, some of the former cultures were returned to TABLE

OF

59

FIBItOBI,ASW

medium with full serum, while the rest were kept on medium with lipid-depleted serum. Replicate cultures were harvested on Days 0, 3, and 5 for fluorescence-polarization measurements at 25°C. The results shown in Fig. 2 indicate that a S-day exposure to medium deficient in lipoprotein-bound cholesterol was sufficient to increase the membrane-lipid fluidity of the control cells (i.e., produce a decrease in the p values) to a level equivalent to that of the FH cells grown in medium with full serum and that this effect was fully reversible within 2 days. The mutant fibroblasts, however, exhibited the same elevated membrane-lipid fluidity regardless of the presence or absence of extracellular lipoprotein-bound cholesterol. This experiment was repeated in abbreviated form with comparable results (not shown). Effect of free cholesterol not bound to lipoproteins on membrane fluidity of fibroblasts. Skin fibroblasts derived from patients homozygous for FH are incapable of binding and taking up extracellular LDL (1-11). Thus, when the only exogenous source of sterol is that which is bound to IV

STATKSTIC‘AI. COMPARKSON OF p VAIJIES ORTAINW WITH NORMAI. (LINE 12) ANTI HYI’F:I~(:HOI.E:S.~~~I~OI.EMIC (LINE 5) CEI.I.S APTFX E:XPOSIII~E TO SERUM-FKBF: MEDIUM WITH OR WITIIOIJT ADI)EI) CIKKFSTEIWI.~ Line

Cholesterol concentration (&ml)

5 5

0 50

5

100

12

0

12

50

Meanp

(df”

value

= 4, t’

(df = 4, t = (df=

4,

(df = 5, (df= 5, 12

t

=

t= t=

100 (df = 5, t = (df= 5, t =

(X lo”) + SE

____

197 + 1.76 (3) 251 f 3.51 (3) = 13.7, Y < 0.001)” 252 -c 6.12 (3) 8.53, 0.01 > P > 0.001)” 248 rt 2.96 (3) 14.6, P < 0.001)” 276 z?z 5.06 (4) 4.88, 0.01 > P > 0.001)” 4.10, 0.01 > P > 0.001)’ 292 + 12.0 (4) 3.62, 0.02 > P > 0.01)” 3.02, 0.05 > P > 0.02)”

” After preincubation in serum-free medium, replicate cultures of fibroblasts were exposed to serum-free medium with or without added cholesterol as described under Materials and Methods. Each p value represents the mean k SE for the combined data from two separate experiments; the number of cultures per experimental condition is shown in parentheses. Statistical comparisons were made between the data by the Student t test. ‘Degrees of freedom = (nl - 1) + (n2 - 1). ’ From Student t test. ” Experimental vs control values with same cell line. ’ Line 5 vs line 12 at same cholesterol concentration.

60

HAGGEH’I’Y

serum proteins, these mutant cells contain less cholesterol than cells from normal individuals (20, 21). This difference in sterol content is seen in spite of the high rate of cholesterogenesis in the homozygous fibroblasts probably because these cells, like heterozygous leukocytes of the same genotype (19), leak cellular sterols into the surrounding medium at an enhanced rate. Since changes in cellular cholesterol levels were responsible for alterations in the membrane-lipid viscosity of mouse lymphocytes and lymphoma cells (41, 42), it was conceivable that the decreased sterol content of the FH cells expected in the presence of medium supplemented with native serum could have been the cause of their elevated membrane-lipid fluidity relative to the values seen in normal cells. In order to test this possibility, cultures of normal and hypercholesterolemic cells were exposed to suspensions of cholesterol in serum-free medium. Under these conditions, the mutant cells were expected to accumulate sterol, since it was shown previously that exogenous cholesterol not bound to lipoproteins was taken up by both normal and hypercholesterolemic fibroblasts (6-8, 20, 43,44) and because neither leukocytes (19) nor hepatocytes4 could lose cholesterol in vitro in the absence of a sterol acceptor in the medium (40). As is evident from the data in Table IV, the delivery of exogenous cholesterol to the hypercholesterolemic cells by this means increased the fluorescence-polarization values (lowered their membrane-lipid fluidity) up to those seen with normal cells incubated in the serum-free medium alone. Exposure of the normal cells to the sterolsupplemented medium caused an even further increase in their p values. DISCUSSION

The experiments described support the idea that homozygous FH fibroblasts have an abnormal cellular membrane structure, as manifested by a greater than normal membrane-lipid fluidity. This abnormality was also associated with a lower than normal concentration of cellular cholesterol, 4 Edwards, unpublished

P. A., Fogelman, observation.

A. M., and Popjik,

G.,

ET AL

and the addition to the culture medium of cholesterol not bound to lipoprotein-a manipulation known to increase the cholesterol content of FH fibroblasts (20)decreased the membrane-lipid fluidity of these cells to values found for normal cells. Growth of normal cells in medium with lipid-depleted or lipoprotein-deficient serum resulted in an induction of HMG-CoA reductase (2, 3, lo-15), an effect which, in turn, was preceded-at least in leukocytes (19) and hepatocytes (42)-by an efflux of cholesterol from the cells. Thus, the increased fluidity of the membrane lipids of normal fibroblasts upon transfer to medium with lipid-depleted serum and the restoration of the fluidity after subsequent growth in medium with full serum may be attributed to depletion and replenishment, respectively, of the cholesterol content of their cellular membranes. These lipid compositional changes in the normal cells and the accompanying alterations of their membrane-lipid fluidity occurring under such experimental conditions are undoubtedly mediated by specific receptors for cholesterol-laden LDL, as demonstrated by the many elegant experiments of Goldstein and Brown. Moreover, our observation that similar manipulations of the FH libroblasts caused no changes in membrane fluidity (Fig. 2) is in harmony with the finding by those authors that fibroblasts of individuals with the receptor-negative form of homozygous FH cannot bind and metabolize LDL. Findings by Chatterjee et al. provide further evidence for the involvement of cellular cholesterol, rather than of other lipids, in the modulation of membrane-lipid fluidity in these experiments (21). Analysis of the glycosphingolipid (GSL), phospholipid (PL), triglyceride (TG), and cholesterol ester (CE) contents of skin fibroblasts revealed the following differences between cells from normal and FH individuals: (i) After growth in medium with full serum, the mutant cells contained much more GSL, PL, and TG and less CE than the normal cells (21). (ii) Upon transfer of the mutant cells to lipoprotein-deficient serum, their GSL, PL, and TG contents markedly decreased to near-normal levels, whereas their CE content remained unchanged (21).

MEMBRANE-LIPID

FLUIDITY

Under similar conditions in our experiments, the membrane-lipid fluidity of these cells did not change either. (iii) Transfer of the normal fibroblasts to lipoprot&n-deficient serum decreased their CE levels but did not affect the levels of the other lipid classes (Zl), wbile the analogous manipulation in our experiments increased the membrane-lipid fluidity of these cells (Fig. 2). Thus, our observations and the findings of Chatterjee et al. taken together provide strong evidence that differences in membrane-lipid fluidity between individual fibroblast lines and alterations in this parameter seen under various experimental conditions cannot be explained by differences or changes in the cellular content of GSL, PL, or TG; they suggest instead that cellular cholesterol content was the main determinant of membrane-lipid fluidity under our experimental conditions and, furthermore, that elevations in this latter parameter in fibroblasts from FH individuals are one additional phenotypic manifestion of a defect in membrane structure and function that may be associated with the abnormality of cholesterol metabolism in FH. ACKNOWLEDGMENTS This research was supported in part by USPHS Grants HD-06576, HL-12745, and HL-18016 from the NICHD and Gralnts 521 IG2 and 574 from the American Heart Association, Greater Los Angeles Affiliate. The authors s, P. A., ANI) PoP.J.&K, G. (1977) J. Biol. Chem. 252, 644-651. 20. BI~OWN, M. S., FAUST, J. R., AND GOl.rXTF.IN, J. L. (1975) J. Clin. Znuest. 55, 783-793. 21. CHATTEI~JEE, S., SEKEI~KF., C. S., AND KwITEltovIcH, P. 0. (1976) Proc. Nat. Acad. Sci. USA 73, 4339-4343. 22. Ho, Y. K., FAUST, J. R., BII.HEIMEH, D. W., BI~OWN, M. S., AND GOI.IJSTF:IN, J. L. (1977) J. Exp. Med. 145, 1531-1549. 23. ESKO, J. D., GII.MOI~E, J. R., AND GI.ASEH, M. (1977) Biochemistry 16, 1881-1890. 24. STFXN, O., WEINSTEIN, D. B., STFXN, Y., AND STEINREHC., D. (1976) Proc. Nat. Acad. Sci. USA 73, 14-18. G. E. (1975) in Microbiology 1975 (Schles25. KENNY, singer, D., ed.), pp. 32-36, American Society for Microbiology, Washington, D.C. 26. HAGGEHTY, D. F., YoIIN~, P. L., Po~.J.&K, G., AND CAI~NF.S, W. H. (1973) J. Biol. Chem. 248, 223-232. 27 HA~GEI~TY, D. F., YOIINC:, P. L., AND BIJESE, J. V. (1974) Develop. Biol. 40, 16-23. 28. SHINITZKI, M., ANr) INRAH, M. (1974) J. Mol. Biol. 85, 603-615.

62

HAGGEHTY

29. SHINITXKI, M., ANIIBAI

Fluorescence-polarization measurements on normal and mutant human skin fibroblasts.

ARCHIVES Vol. OF BIOCHEMISTRY 189, No. 1, July, AND pp. 51-62, Fluorescence-Polarization DONALD Departments University BIOIBHYSICS 1978 Meas...
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