Influence of ethanol on fatty acid composition of phospholipids in cultured neurons

Vol. 122, No. 2, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 516-521

July 31, 1984

INFLUENCE

OF ETHANOL

ON FATTY A C I D C O M P O S I T I O N IN C U L T U R E D NEURONS

M. Morrisson, Department

P.A.

Wilce

and B.C.

OF P H O S P H O L I P I D S

Shanley

of B i o c h e m i s t r y , U n i v e r s i t y of Q u e e n s l a n d , St. Lucia. 4067. A u s t r a l i a

Received June 18, 1984 A n i m a l s c h r o n i c a l l y e x p o s e d to e t h a n o l show changes in neural m e m b r a n e lipids which may underlie the d e v e l o p m e n t of t o l e r a n c e and p h y s i c a l d e p e n d e n c e . The o b j e c t of this study was to i n v e s t i g a t e changes in the fatty acid c o m p o s i t i o n of neuronal p h o s p h o l i p i d s c u l t u r e d in the p r e s e n c e of ethanol (55 or ii0 mM) for periods up to 7 days. Decreases were o b s e r v e d in the p e r c e n t a g e of individual and total s a t u r a t e d fatty acids, while the double b o n d index: total s a t u r a t e d fatty acid ratio, increased. These changes do not s u p p o r t the h y p o t h e s i s that neural m e m b r a n e lipid c o m p o s i t i o n changes to c o u n t e r a c t the f l u i d i z i n g action of ethanol.

It has

long been

the central physical

nervous

state

suspected

s y s t e m are m e d i a t e d

and chemical

A number of studies

biological

membranes

exposure

membrane

tolerance

which

dependence

is no c o n c l u s i v e

abovementioned

membrane

e f f e c t of ethanol

perturbation a m e t h o d of

of

Abbreviations

evidence

on nerve

lipid m e t a b o l i s m .

used:

between

All rights of reproductton m any form reserved.

thought

516

the

plasma can d i s r u p t

(1-3).

to changes

in

to r e p r e s e n t and w h i c h may

associated

with

to date

as to whether

occurring

the

{n ~{uo reflect

a

cells or a g e n e r a l i z e d The use of cell culture direct

DBI, double bond acids

0006-291X/84 $1.50 Copyright © 1984 by Academtc Press, lnc

on

on

(4-6).

lipid changes

distinguishing

fluidity

leads

function

effects

that ethanol

membrane

are

of ethanol

the neuronal

effect of ethanol,

in neuronal

and p h y s i c a l

of

shown

to ethanol

to the fluidizing

There

direct

of animals

alterations

have

increase

lipid c o m p o s i t i o n

adaptation mediate

and

through

composition

membrane.

Chronic

that the actions

and

index;

indirect

SFA,

affords

effects

saturated

of

fatty

Voh 122, No. 2, 1984

ethanol shown

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on c e l l u l a r

that c h r o n i c

ethanol

in c u l t u r e

properties.

function

of dorsal

produces

changes

electrophysiological composition,

model

system.

ethanol

on

cultures

in e l e c t r i c to expect

are d e p e d e n t

the latter

fatty

of neurons

MATERIALS

changes

and Edwards

root g a n g l i o n

seem r e a s o n a b l e

upon

study we r e p o r t

acid c o m p o s i t o n obtained

(8) have neurons

to

membrane that,

if these

altered membrane

should be d i s c e r n a b l e

In the p r e s e n t

the

Scott

exposure

It w o u l d

lipid

(7).

the

in such

influence

of p h o s p h o l i p i d s

a of

in p r i m a r y

from mice.

AND METHODS

F o u r t e e n - d a y - o l d Q u a c k e n b u s h mice embryos were used to e s t a b l i s h p r i m a r y neuronal cultures. Embryos were d e c a p i t a t e d and the whole b r a i n r e m o v e d and g e n t l y h o m o g e n i z e d by a s p i r a t i o n through a p a s t e u r pipette. After removal of u n h o m o g e n i z e d debris, cells were s u s p e n d e d in m e d i u m c o m p r i s i n g RMPI 1640 (GIBCO) and 10% foetal calf serum, and p l a t e d o u t in f l a t - s i d e d flasks at a d e n s i t y of 5.7-11.5 x 104 cells per cm 2. Cultures were m a i n t a i n e d at 37°C in an a t m o s p h e r e of 5% C O 2 : 9 5 % air. After 4 days in culture, m e d i u m c o n t a i n i n g cytosine a r a b i n o s i d e (5 x 10-5M) was s u b s t i t u t e d to p r e v e n t glial cell o v e r g r o w t h and p r o m o t e neuronal d i f f e r e n t i a t i o n (9). Four days later this was c h a n g e d for fresh m e d i u m w i t h or w i t h o u t ethanol. After a further 1 to 7 days in culture, cells were h a r v e s t e d with trysin solution, washed, h o m o g e n i z e d and e x t r a c t e d with c h l o r o f o r m : methanol: :2:1.

The c h l o r o f o r m - m e t h a n o l e x t r a c t was e v a p o r a t e d to dryness under nitrogen, r e d i s s o l v e d in a small volume of c h l o r o f o r m : m e t h a n o l : : l : l and c h r o m a t o g r a p h e d on thin layer plates (Silica gel H : F l o r i s i l R : :12.5:1) using p e t r o l e u m s p i r i t : d i e t h y l ether: g l a c i a l acetic a c i d : : 9 0 : 1 0 : 2 c o n t a i n i n g 1% (v/v) b u t y l a t e d hydroxytoluene. Lipid classes were d e t e c t e d by e x p o s u r e to iodine vapour and i d e n t i f i e d by c o m p a r i s o n of Rf values with know n s t a n d a r d s . The b a n d c o r r e s p o n d i n g to total p h o s p h o l i p i d s was s c r a p e d off the plates and e l u t e d with c h l o r o f o r m : methanol::l:l. Fatty acid methyl esters were p r o d u c e d and a n a l y s e d by gas liquid c h r o m a t o g r a p h y as d e s c r i b e d p r e v i o u s l y (7).

RESULTS

AND

DISCUSSION

Control significant the course obtained

cultures change

of

grown

in the absence

in p h o s p h o l i p i d

the e x p e r i m e n t .

by g r o w t h of neurons

showed

fatty acid c o m p o s i t i o n

Tables

i and

in 55 mM and

517

of ethanol

2 show

no

during

the results

ii0 mM e t h a n o l

Vol. 122, No. 2, 1 9 8 4

Table

Fatty

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i: Fatty acid composition of neuronal p h o s p h o l i p i d s following culture,in ethanol (55 mM)

acid

Time(days) 1 3 (n = 3) (n = 5)

0 (n = 5)

16:0

18.47,0.91

16:1

5.84±0.72

15.14,0.47"

7 (n = 2)

18.18a0.44

17.88~1.58

9.81~1.94

12.46,8.04

3.11e0.06"

18:0

18.89~1.31

13.03,0.71"*

14.74m0.44

12.88~0.93"

18:1

22.82*0.57

15.56,0.61"**

23.83~1.79

20.06~i.09 1.20~0.40

18:2

1.53,0.42

0.81±0.07

1.25~0.22

18:3+20:1

1.84±0.90

0.27±0.09

0.69~0.ii

N.D°

20:2

3.81,1.25

1.77±0.51

4.45~0.49

22:1+20:3

8.32±1.56

9.05~0.85

6.02±1.03

9.71~0.64

20:4

1.25,0.30

0.76±0.09

1.37~0.14

1.37~0.02

24:1

1.82±0.26

1.93±0.12

1.56~0.19

1.71~0.22

22:6

1.01±0.23

1.84±0.09

1.22~0.15

1.77~0.29

12.08±0.80

17.19±4.68

iI.84~0.88

ii.93~1.67

DBI

62.38±3.80

49.16±1.61"

66.11~3.22

62.41~8.40

ZSFA

37.36±1.60

28.17,0.85"**

32.92~0.62"

30.71~1.83

ULC

DBI:ZSFA

1.67±0.12

1.75±0.08

N.D.

2.01~0.10

2.03~0.30

n = number of experiments, each performed in triplicate Results are mean percentage ± S.E.M. Statistical significance of differences between 0 days and I to 7 days of exposure to ethanol was c a l c u l a t e d using Student's ttest. * 0.05 > p > 0.02 ** 0.02 > p > 0.01 *** 0.01 > p > 0.001 N.D. = not detected DBI = double bond index SFA = total % saturated fatty ULC = u n i d e n t i f i e d long chain

respectively, in

the

were

for

percentage

still

of

evident

differences

were

ethanol

for

and

unsaturated statistically

periods

at

acids fatty

up

saturated the

fatty

at

acids,

significant

7 days. fatty

termination

statistically 18:0

to

acids

55 m M 16:1 at

Decreases

acids of

the

significant ethanol showed 7 days

518

(p an

(16:0,

observed

18:0).

These

experiment.

for < 0.05)

increase

culture

were

in

16:0

at

ii0

Among which ii0 m M

The mM

the was

ethanol.

Vol. 122, No. 2, 1 9 8 4

Table

Fatty

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2: Fatty acid c o m p o s i t i o n of neuronal p h o s p h o l i p i d s following culture in ethanol (ii0 mM) Time 1 (n = 4)

acid 0 (n = 5)

(days) 3 (n = 4)

7 (n = 2)

16:0

18.47~0. 91

16.67~i.13

14.64±1.24

16:1

5.84~0. 72

6.80~1.51

6.44±1.40

17.49ei.92

13.79±0.73"

18:0

18.89~i. 31

19.72±1.99

19.15±2.63

13.41±0.50

18:1

22.82±0. 57

19.88m2.31

29.00±4.44

17.63~0.73

18:2

1.53~0. 42

1.74±0.40

1.96±0.58

1.52±0.31

18:3+20:1

1.84±0

90

0.86±0.19

0.52±0.04

0.48±0.11

20:2

3.81±1

25

2.94±0.59

2.76±0.59

2.42±0.77

22:1+20:3

8.32±1

56

8.26±1.12

7.85±1.47

9.64~0.25

20:4

1.25±0

30

1.08±0.14

0.71±0.27

24:1

1.82±0

26

1.37±0.15

1.56±0.33

22:6

1.01±0

23

1.04±0.22

1.97±0.65

1.68±0.09

12.08±0

80

14.67±2.30

13.73±2.07

16.43±1.94

ULC

N.D. 1.63±0.11

DBI

62.38~3.80

57.03±3.61

69.47±6.56

63.83±2.71

ZSFA

37.36±1.10

36.39±2.29

33.79±2.91

27.20±0.88

1.67±0.12

1.57±0.14

2.06±0.26

2.35±0.13

DBI:ZSFA

n = number of e x p e r i m e n t s , each p e r f o r m e d in triplicate R e s u l t s are m e a n p e r c e n t a g e ± S.E.M. S t a t i s t i c a l s i g n i f i c a n c e of d i f f e r e n c e s b e t w e e n 0 days and 1 to 7 days of e x p o s u r e to e t h a n o l was c a l c u l a t e d using S t u d e n t ' s ttest. * 0.05 > p > 0.02 ** 0.02 > p > 0.01 *** 0.01 > p > 0.001 N.D. : not d e t e c t e d DBI = double b o n d index SFA = total % s a t u r a t e d fatty acids ULC = u n i d e n t i f i e d long chain fatty acids

The degree

double

of

unsaturation

~7(percentage double

was

not

in

followed

results

of

bonds).

reduction

bond

were

each Cells

the by

DBI

of

(DBI) the

is

fatty

unsaturated grown after

a return

obtained

statistically

index

in

with

55 m M

the

(4).

acid

for

one

x

ethanol,

the

number

showed day

but

overall

It r e p r e s e n t s the

level

(p > 0 . 0 5 ) .

519

of

ethanol

control

110 m M

significant

acids

fatty

exposure

to

a measure

(p

by

its

a significant < 0.05).

3 days.

the

of

the

This

Similar

difference

was

Vol. 122, No. 2, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The p r o p o r t i o n are s a t u r a t e d intervals

ethanol

the

grown

was m a i n t a i n e d (p < 0.02).

concentrations significant

total p h o s p h o l i p i d

(SFA) was s i g n i f i c a n t l y

in cells

decrease

of

Finally,

of ethanol.

for cells

decreased

in 55 mM and at 7 days

grown

acids

for cells g r o w n DBI:SFA

increase

which

at various

ii0 mM ethanol.

the ratio

This

fatty

time

The

in Ii0 mM

increased

at both

was s t a t i s t i c a l l y

in ii0 mM ethanol

for

7 days

(p <

0.O5).

Our r e s u l t s ethanol of

promotes

HeLa

(7).

administration Littleton

this

of

workers

together

have

synaptosomal ethanol

On

occurred

found plasma

membrane

with c u l t u r e d in vivo

findings.

in the r e l a t i v e

In a later receiving

administration

study

they

found

a high s a t u r a t e d (i0).

Other

in the p h o s p h o l i p i d

fatty acids of

in rats

treated

chronically

with

(ii, 12).

theoretical

fluidizing

phospholipid

in the relative acyl groups

do not s u p p o r t to the view following

grounds

an adaptive

e f f e c t of ethanol on neural

increase

dietary

no change

Similar

employing

different

in animals

with ethanol

laboratory

an increase

fatty acids.

to

degree of s a t u r a t i o n

studies

have y i e l d e d

only

in culture

phospholipids.

in this

hand,

(4) r e p o r t e d

saturated

increase

fat diet,

On the o t h e r

and John

proportion

membrane

previously

of ethanol

of neurons

in the relative

of neural

were o b t a i n e d

cells

that e x p o s u r e

a decrease

the acyl g r o u p s

results

an

indicate

this

that

chronic

factors,

proportions

(13).

hypothesis.

the p r e s u m e d ethanol

membranes

should

of

of

changes

seen

study

in vivo

dependent

lipoprotein

fluidity.

520

the p r e s e n t

they lend credence

may be nore

in p l a s m a

involve

saturated:unsaturated

findings

adaptive

of membrane

to the

Furthermore,

exposure,

and changes

than on h o m e o s t a s i s

The

response

upon

metabolism,

Vol. 122, No. 2, 1 9 8 4

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13.

Grenell, R.G. (1975) Adv. Exp. Med. Biol. 59, 11-22. Chin, J.H. and Goldstein, D.B. (1977) MOI. Pharmacol. 13, 435-441. Harris, R.A. and Schroeder, F. (1981) Mol. Pharmacol. 20, 128-137. Littleton, J.M. and John, G.R. (1977) J. Pharm. Pharmac. 29, 579-580. Chin, J.H., Parsons, L.M. and Goldstein, D.B. (1978) Biochim. Biophys. Acta, 513, 358-363. Johnson, D.A., Lee, N.M., Cooke, R. and Loh, H. (1979). Mol. Pharmacol. 15, 739-746. Keegan, R., Wilce, P.A., Ruczkal-Pietrzak, E. and Shanley, B.C.(1983) Biochem. Biophys. Res. Commun. 114, 985-990. Scott, B.S. and Edwards, B.A.V. (1981) J. Neurobiol. 12, 379-390. Dambergs, R., Leah, J. and Kidson, C. (1978) Exp. Neurol. 59, 296-303. John, G.R., Littleton, J.M. and Jones, P.A. (1980) Life Sci. 27, 545-555. Wing, D.R., Harvey, D.J., Hughes, J., Dunbar, P.G., McPherson, K.A. and Paton, W.D.M. (1982) Biochem. Pharmacol. 31, 3431-3439. Crews, F.T., Majchrowicz, E. and Meeks, R. (1983) Psychopharmacol. 81, 208-213. Hill, M.W. and Bangham, A.D. (1975) Adv. Exp. Med. Biol. 59, 1-9.

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