The effect of culture conditions on the fluidity of mouse neuroblastoma membranes as estimated by spin label studies

Printed in Sweden Copyright @ 1977 by Academic Press. Inc. All rights ofreproduction in any form reserved ISSN 00144827

Experimental

THE EFFECT FLUIDITY

Cell Research 109 (1977) 381-387

OF CULTURE

OF MOUSE ESTIMATED

CONDITIONS

NEUROBLASTOMA BY SPIN LABEL

ON THE

MEMBRANES

AS

STUDIES

Wm. G. STRUVE, R. M. ARNESON, J. E. CHENEVEY and Ch. K. CARTWRIGHT Department of Biochemistry, The University of Tennessee Centerfor the Health Sciences, Memphis, TN 38163, USA

SUMMARY Gas chromatography and spin labeling were used to estimate the composition and fluidity of the lipid acyl chains in intact cell membranes of mouse Cl300 neuroblastoma. Neurite formation and an increase in the specific activity of acetylcholinesterase are induced in this cell line by the removal of serum from the culture medium for one day prior to harvesting the cells. The fatty acid composition of induced cells was not significantly different from that of the non-induced controls. Each of the three fatty acid spin labels used in this study indicate that the membranes of induced cells are slightly more fluid than the membranes of control cells. This difference in fluiditv annears to be a result of a reduction in the progressive decrease in fluidity occurring during gro-wth in serum. A four-fold reduction in cell viability had no effect on the measured bilayer fluidity. Thus the changes in fluidity appear to be due to the presence or absence of serum rather than to cell type, age, or viability.

Mouse neuroblastoma cells (C 1300) growing logarithmically in culture can be induced by a variety of treatments to undergo morphological and biochemical differentiation to non-dividing cells with neuron-like properties [l]. This cell line has been used extensively in the study of neurochemistry, neoplasia and differentiation. Recently attention has been focused on changes in the plasma membrane during differentiation. Among these changes are increased acetylcholinesterase activity [2], increased number of acetylcholine receptors per cell [3], changes in the electrophysiological properties of the cell membrane [4], increased sensitivity of adenyl cyclase to dopamine and norepinephrine [5], and the appearance of neural antigens [6]. The most striking aspect of morphologi25-771804

cal differentiation in neuroblastoma cells is the formation of axon-like neurites [7]. In this paper, the terms log cells and neurite cells are used to designate respectively, the logarithmically growing cells and neurite-bearing cells. The term differentiation is used in this paper and throughout the literature to designate the transformation between these two types of cells. A discussion of the relationships between such transformations and more comprehensive views of differentiation may be found in Foulds’ monograph [8]. Changes in the biophysical properties of cell membranes may be estimated by the use of spin labeled fatty acids. Spin labeling is an established technique suited to the study of living cells (for an extensive review see [9]). In addition, because the conExp Cell Res 109 (1977)

382

Struve et al.

centration of naturally occurring free radical is too low to be observed by our methods only the added label contributes to the observed Electron Paramagnetic Resonance (EPR) spectrum. More importantly, the work of McConnell [lo] had demonstrated the reproducibility of changes as small as 1% in the order parameter of spin labeled fatty acids in cell membranes. Although sensitive, selective and theoretically well understood, this technique is not sufficient by itself to determine the underlying causes of changes in membrane properties. Use of fatty acid spin labels is limited by perturbations of the membrane due to the presence of fatty acid and bulky nitroxide moiety. Differences in the membrane fluidity between contact inhibited and transformed mouse tibroblast 3T3 cells as measured by the spin label technique have been reported [ 111. Considerable doubt, however, has been cast on those results since the spin label used in those studies is now knotin to be mixture of three compounds [12]. In addition, Gaffney, using two fatty acid spin labels, was unable to detect any significant differences in the membrane fluidity of normal and transformed mouse fibroblasts [ 131. To more fully delineate the differences between the membranes of log and neurite cells, we have measured their fluidity by the spin label technique which records the flexibility of the hydrocarbon chains of the membrane lipids. The differences we observe are discussed in relation to cell growth and fatty acid composition. MATERIALS

AND METHODS

Culture and analysis of neuroblastoma cells Mouse neuroblastoma C1300, clone N-18, were obtained from Dr Marshall Nirenberg. Cells were grown Exp Cell Res 109 (1977)

without antibiotics in Dulbecco’s modification of Eagle’s medium (Gibco Cat. No. H-16) in 10% CO,, 90% air with 10% fetal calf serum (lot no. 90129 Microbiological Associates). The cells were subcultured everv 6 davs usinn one-fiftieth of the cells from one of the-original flask; to inoculate each new flask. All cell culture was carried out in 75 cm2 flasks (Falcon Co.). The cells were fed every 2 days with 15 ml of medium. Cell cultures used in these experiments were shown to be free of mycoplasma contamination by the method of McGarrity [ 141. Cells were harvested for subculture or experimental use as follows. The medium was decanted and each flask washed gently with 5 ml of Blume’s modified D, solution [2]. Both medium and wash solutions were discarded. The cells were removed from each flask with 5 ml portions of modified D, Versene solution TlSl. Versene solution and modified D, in that order. The. flasks were incubated at 37°C with the first 5 ml uortion of Versene solution. After centrifugation at 400 g for 10 min the cell pellet was resuspended in 40 ml of modified D,. A sample of the isolated cells was counted in a hemocytometer and cell viability was determined by the dye exclusion test using trypan blue [16]. The fairly low percent viabilities are those expected for cells isolated in balanced saline solutions [ 171. Acetylcholinesterase activity was measured by the method of Ellman [18] as modified by Schubert and co-workers f191. One unit of activitv is defined as 1.O nmole of product formed per min. Cells were transformed from log to neurite cells by incubation in culture medium without fetal calf serum for 24 h.

Spin label studies CHsW-Lh~~HACOOH 0

H&Z-

(1)

7”’

C-CHI CH,

The three fatty acid spin labels I@, n) having (m. n) =(12, 3), (5, lo), and (1, 14), obtained from Syva Associates, Palo Alto, Ca, were used without further purification. Cells dispersed in modified D, solution were sedimented by centrifugation at 400 g for 10 min. A 100 ~1 sample of the pellet containing about 10’ cells was withdrawn, transferred to a 10x75 mm culture tube containing 7.5 nmoles of the spin labeled fatty acid, and gently mixed for 10 min. The remainder of the pellet was gently resuspended in the original supernatant and held at room temperature between samplings. The spin labeled cells were drawn into a 100 ul disnosable Dinette which was then sealed. The pipette was placed ;n the cavity of a Varian model 4502 EPR spectrometer. All spectra were recorded using less than 10 mW microwave power at 37+ O.Ol”C. Considerable reduction in the amplitude of the signal was observed during repeated measurements of the same sample over a 2 h period, but no significant change in the order parameter resulted from these

Neuroblastoma

membrane fluidity

383

Table 1. Fatty acid composition

changes in amplitude (see fig. I). The order parameters were calculated from the recorded spectra by the method of Gaffney [20], using a value of 1.66 for the constant: (T,,+T,,)/(T,,-(T,,+T,,)/2).

of neuroblastoma cells 5 days after subculture

Fatty acid analysis

Fatty acids

A

B

C

16:0 16:l 18:0 18: 1 18:2 20:o 18: 3/20 : 1 20:3a 20:3b 20:4 22:4 22 : 5 22 : 6

25.5 1.4 14.5 37.5 1.8 1.1 2.9 1.1 1.1 6.6

22.6 1.6 14.2 35.1 1.5 1.1 2.9 1.1 1.2 6.1 2.1 1.8 2.4

24.0 6.5 14.4 31.1 1.1 1.1 3.2 1.3 1.0 4.9 2.1 1.5 1.1

Neuroblastoma linids were extracted bv the method of Bligh & Dyer (211. Transesteritication was carried out in anhvdrous methanol-HClr221 and the resulting methyl esters were purified by thin layer chromategraphy on silica gel G [23]. Isothermal gas liquid chromatography was performed on a Hewlett-Packard 402 Gas Chromatograph with a 6 ftx3/16 in glass column using 10% EGGS-X on Gas-Chrom P (Applied Science Laboratories) as the stationary phase. The operating temperatures were 200 and 220°C. Methyl lignocerate was used as an internal standard. In general, the procedures referred to above were conducted in the presence of 10 mg% 2,6-di-tert-butyl-p-cresol (BHT) (Sigma Chemical Co.), and under nitrogen when feasible. Commercially available standards (Applied Science Laboratories) were used to determine the identity of the gas chromatographic peaks. The identity of these peaks was confirmed by analysis on a Finnigan 3200 Gas Chromatograph-Mass Spectrometer using 3 % EGGS-X on Gas Chrom Q as the stationary phase. Methane was used both as carrier gas and ionizing reagent.

RESULTS

Composition in area percent

T T T

Each column is the mean of data from two separate extractions of neuroblastoma cells. Column A, cells cultured three months prior to the single passage represented in columns B and C. Serum was withdrawn from the cells represented in C one day prior to harvest. T, a fatty acid could be detected but not accurately determined.

were 9.7 Ulmg, 8.6 Ulmg and 17 Ulmg, respectively.

Changes occurring in neuroblastoma cultures upon withdrawal of serum

Fatty acid composition cells

The decline in cell division, the formation of neurites and the increase in acetylcholinesterase specific activity we observe upon withdrawal of serum from neuroblastoma cultures are essentially the same as those reported by other workers [2]. Acetylcholinesterase activity is used routinely as an index of differentiation in neuroblastoma cells. Removal of serum for one day any time after the second day of culture led to an approximate doubling of acetylcholinesterase activity compared with controls incubated with serum. For example, of the cells used for the fatty acid analyses in table 1, only those in col. C were incubated without serum for one day prior to harvest. The specific activities of acetylcholinesterase for the cells of columns A, B and C

Table I indicates that the fatty acid composition of neuroblastoma cells was not affected by a one-day withdrawal of serum nor were changes observed with 15 passages after three months in culture. A comparison of data groups A, B and C by an analysis of variance found no difference between them (P>O.O5). Methyl myristate was detected by mass spectrometry but could not be quantitated by gas chromatography due to the presence of the antioxidant BHT. As table 1 indicates, the methyl esters of linolenic and eicosenoic acids co-migrate in the chromatographic system employed. The isomers responsible for peaks 20: 3a and 20: 3b have not been identified. In addition to the fatty acids given in table 1, traces of fatty

of neuroblastoma

E-t{> Cell RPS IOY (IY77)

384

Struve et al. Fig. 1. Typical EPR spectrum of

label I (12, 3) incorporated into neuroblastoma log cells harvested 5 days after subculture. Cells were suspended in modified Di buffer and all spectra were recorded at 37+ O.Ol”C. The microwave power, modulation amplitude, scan time, scan range and filter time constant are 5 mW. IG. 10 min. 100e and 0.3 set, respectively. Pachjrace represents a successive decrease in spectral intensity due to spin label reduction. Arrows indicate hyperfine extrema.

acids having the formulas 22 : 1 and 22 : 3 can be detected by gas chromatographymass spectrometry. Membrane fluidity, viability and culture-induced differentiation Table 2 shows the relationship between cell viability and the spin label order parameter over a 9 h period after harvesting log cells. During that time, the viability dropped by more than 70% whereas the order parameter decreased less than 1%. Table 3 shows the differences in order parameters of three different spin labels between log cells and neurite cells. Also shown in this table is an increase in the order parameter with the total number of days of growth after subculture. In addition, the expected decreases [9, lo] in order

2. Typical EPR spectra of label I (5, IO) incorporated into log cells (solid line) or neurite cells (broken line) harvested 3 days after subculture. Other conditions were similar to those in fig. I.

Fig

Exp CrllRrsl09

(1977)

parameter as the number of methylene groups between the carboxyl and the nitroxyl increases from 3-10 to 14 is shown for both log and neurite cells in table 3. Some of the data of table 3 has been plotted in fig. 3 to clarify the effects of serum removal, time after subculture, and type of spin label used to measure the order parameter. Typical EPR spectra for several conditions are shown in fig. 2.

Table 2. Dependence of membrane fluidity as measured by the spin label order parameter on cell viability as measured by dye exclusion Time (hours)

Viability (So)

T,‘,(‘3)

TL(G)

Order parameter

I.0 2.2 2.3 3.3 4.8 9.3

64 57 56 51 42 17

22.20 22.20 22.20 22.12 22.11 22.10

9.40 9.42 9.42 9.40 9.42 9.40

0.472 0.471 0.471 0.470 0.468 0.469

Mean order parameter=0.470 (S.D.=0.0015). Log cells were transferred from growth medium to modified D, buffer and incubated for the time indicated at room temperature. Spin label I (12, 3) was used in the determination of T,',and T; which are equal to the separations (in Gauss) of the outer and inner spectral extrema, respectively.

Neuroblastoma

membrane fluidity

385

Table 3. Difference

in membrane fluidity as measured by the spin label order parameter resulting from differentiation induced by removal of serum for one day Order parameter Label

Days

Log cells 0’)

Neurite cells 0’)

Difference

I(12, 3) I(12, 3) 1(12, 3) I(5, IO) I(5. IO) I(5, IO) ICI, 14)

3 4 5 3 4 5 4

0.455 (2) 0.474 (2) 0.487 (2) 0.169 (1) 0.191 (2) 0.219 (I) 0.094 (2)

0.432 (1) 0.454 (2) 0.464 (2) 0.153 (2) 0.164 (3) 0.191 (I) 0.080 (2)

-0.023 -0.020 -0.022 -0.015 -0.027 -0.028 -0.014

N. number of independent subcultures grown for the indicated number of days (total). Neurite cells were grown for the last day only in culture medium without serum.

DISCUSSION The fatty acid complement of cultured cells resembles that of the serum employed and removal of serum or serum lipids from the culture medium results in a profound decline in cellular polyunsaturated fatty acids [24]. Such a decline could influence the fluidity of the plasma membrane lipid bilayer [13]. However, the cells used in these experiments were harvested one day after the withdrawal of serum and no change in the fatty acid composition occurs in that time. Although the analyses presented here do not preclude changes in plasma membrane fatty acids, they render such changes unlikely. Using the same neuroblastoma clone (N18) employed in this work, Yavin and co-workers observed no changes in the rates of incorporation of 14C-fatty acids or in their distribution among various lipid fractions for periods up to 48 h after serum withdrawal [25]. That the fatty acid composition of neuroblastoma cells did not change during 3 months in culture suggest a high degree of homogeneity among the cell samples used in this work. Such homogeneity requires adherence to a uniform pattern of subculturing and the use of a single lot of serum for a given series of experiments 1261.

The location or locations of the three spin labels in the neuroblastoma cells used in these studies has not been established. However, the decrease in order parameter from 1(12, 3) to (5, 10) and from I(5, 10) to I(14, 1) is consistent with their localization in lipid bilayers [ 131.In addition, the nitroxide group of spin labels is rapidly reduced to the non-paramagnetic hydroxylamine by components in the cytoplasm of many

0.50 0.45 0.42

:

I,

1 Fig. 3. Abscissa:

I

,

I

,

I,

I

2 3 4 time (days); ordinate:

I

I

5 order para-

meter. Change in the order parameter with time after subculture. The order parameters for label I (12, 2) (upper curves) or label I (5, 10) (lower curves) were measured in log cells (squares) and neurite cells (circles). Some of the data from table 3 are plotted here for clarification. Exp

Cd/

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109 (1977)

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Struve et al.

cells [27], which precludes the observation of signals arising from spin labels located within intracellular membranes. Thus it is likely that the fluidity of the plasma membrane predominates in determining the observed order parameters. Neurite cells have slightly more fluid membranes than do the undifferentiated log cells. One might argue that differentiation leads to an increase in the fluidity of the membranes. An alternative explanation is that growth in the presence of serum results in a progressive decrease in membrane fluidity whereas fluidity does not change with culture in serum-free medium. The latter explanation is supported by comparing the order parameters of log cells grown for 3 and 4 days with neurite cells grown 4 and 5 days since the latter cells are grown for one day in serum-free media (i.e. compare 0.455 with 0.454, and 0.474 with 0.464, and 0.191 with 0.191 in table 3, or circles with squares, fig. 3). We observe a decline in cell division, an increase in the specific activity of acetylcholinesterase, and the formation of neurites, but not a change in fatty acid composition upon the withdrawal of serum for one day. Thus, the progressive decrease in membrane fluidity with the number of days culture with serum is not likely to be due to changes in their fatty acid composition. The serum-induced decrease in membrane fluidity may also be the direct effect of slow incorporation of a component of the serum into neuroblastoma membranes. Such a component would not necessarily need to be incorporated in large amounts. For example, small but reproducible changes in the order parameter and deformability of intact erythrocyte membranes are induced by as little as 10-l’ M prostaglandins E, and E, [lo]. Alternatively, the changes may be due to alteraExp Cell Res 109 (1977)

tion in the ratio of cholesterol to phospholipid in the cell membrane. Uptake (or loss) of cholesterol may occur in the presence of (or absence) of serum. The direct incorporation of a serum component is inconsistent, however, with the small, random fluctuations we see in the order parameter (-tO.O03) with successive passages of this cell line. Whatever leads to the progressive decrease in membrane fluidity must therefore have its effect reversed by dilution of the cells fifty-fold. Perhaps reduction of membrane fluidity is a result of increasing cell density or a change in the membrane protein/lipid ratio. It may be one aspect of the system responsible for the autoregulation of growth in these cells. No relationship was found between the bilayer fluidity of neuroblastoma cell membranes and cell viability as assessed by the dye-exclusion test. There is evidence that dye uptake in this viability test reflects structural changes in the cell membrane [28]. Within the limits of our method of determination, the plasma membrane bilayer does not appear to be affected by these structural changes. It may be possible to distinguish between the relative roles of serum starvation and differentiation in the observed change in order parameter by inducing differentiation by an agent in the presence of serum. Some of the inducing agents (e.g. prostaglandins) are hydrophobic, and might perturb the lipid fluidity directly, so the time course of differentiation and order parameter changes would have to be carefully monitored. We are grateful to Dr Joseph D. Wander for performing the gas chromatographic-mass spectrometric analyses of fatty acid methylesters and to Mr Glen S. Germain for culturing the cells used in these experi-

merits. This work was supported by USPHS Grants CA16927and NS9564 from NIH.

Neuroblastoma membranefluidity REFERENCES I. Prasad, K N, Biol rev 50 (1975) 129. 2. Blume, A, Gilbert, F, Wilson, S, Farber, J, Rosenberg, R & Nirenberg, M, Proc natl acad sci us 67 ( 1970)786. 3. Simantov. R & Sachs, L, Proc natl acad sci US 70 (1973) 29ci2. 4. Chalazonitis, A &Greene, L A, Brain res 72 (1974) 340. 5. Prasad, K N & Gilmer, K N, Proc natl acad sci US 71 (1974) 2525. 6. Akeson, R & Herschman, H, Nature new bio1249 (1974) 620. 7. Augusti-Tocco, G & Sato, G, Proc natl acad sci US64(1969)311. 8. Foulds, L, Neoplastic development, vol. 1, p. 318. Academic Press. New York (1959). 9. Berliner, L J (ed), Spin labeling theory and applications. Academic Press, New York (1976). 10. Kurg, P G, Ramwell, P W & McConnell, H M, Biochem biophys res comm 56 (1974) 478. Il. Barnett, R E, Furcht, L T & Scott, R E, Proc natl acad sci US 71 (1974) 478. 12. - Ibid 72 (1975) 1217. 13. Gaffney, B J, Proc natl acad sci US 72 (1975) 664. 14. McGarrity, G J, Tissue culture association manual l(l975) 113. 15. Paul, J, Cell and tissue culture, 4th edn, p. 218. E & S Livingstone, Edinburgh (1970).

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16. Phillips, I-I J, Tissue culture: methods and applications (ed P F Kruse, Jr & M K Patterson, Jr) p. 406. Academic Press, New York (1973). 17. Phillips, H J & Andrews, R V, Exp cell res 16 (1958) 678. 18. Ellman. 3 L, Courtney, L D, Andreas, V & Featherstone, R M, Biochem pharmacol7 (l%l) 88. 19. Schubert. D, Tarikas, H, Harris, A J 8r Heinemarm, S, Nature new bio1233 (1971) 79. 20. Gaffney, B J, Spin labeling theory and applications (ed L J Berliner) p. 567. Academic Press, New York (1976). 21. Bligh, E G & Dyer, W J, Can j biochem physio137 (1959) 911. 22. Christie, W W, Lipid analysis, p. 88. Pergamon Press, New York (1973). 23. Eng, L F, Lee, Y L, Hayman, RB & Gerstl, B, J lipid res 5 (1964) 128. 24. Bailev. J M & Dunbar, L M._*EXP mol path01 18 _ (1973)‘142. 25. Yavin, E, Yavin, Z & Menkes, J H, J neurochem 24(1975)71. 26. Alovo. V J & Codd. E E, In vitro 10 (1974) 376. 27. St&d, W G. In preparation. 28. Yip, D K & Auersperg, N, In vitro 7 (1972) 323. Received January 24, 1977 Revised version received May 3 1, 1977 . Accepted June 2, 1977

Exp Cell Res 109 (1977)