Choline uptake in neuroblastoma cell cultures: Influence of ionic environment

Pharmacological Research Communications, Vol. 5, No. 4, 1973 CHOLINE

UPTAKE

IN N E U R O B L A S T O M A IONIC

R. Massarelli,

397 : INFLUENCE

CELL CULTURES

OF

ENVIRONMENT A. Ebel 1 and P. Handel

J. C i e s i e l s k i - T r e s k a ,

Centre de N e u r o c h i m i e du CNRS, and Institut de Chimie Facult6 de H6decine, 67085 Strasbourg Cedex, France

Biolozique

i~eceived 6 June 1973

SUMMARY The uptake mouse tion

of choline

neuroblastoma

strongly

cells

ions

also

Incubation inhibited

potassium

incorporation

a strict

relationship

and that

an active transport

The uptake

cells.

between

tissues

(Sung and Johnstone,

Adamic,

1972),

1969; banks,

Green 1968;

1Charg6e

cells

et al.,

1972)

Diamond

de R e c h e r c h e

on the uptake

by

of cho-

out that there

is

and sodium pump is involved

in

cells.

investigated Sanford

or s u b c e l l u l a r

au CNRS.

cesium

in both p r o l i f e r a -

uptake

and Martin,

and Milfay,

was

or at low t e m p e r a -

energy-dependent,

(Hodgkin

with

diffe-

of p o t a s s i u m

It is pointed

1965;

from

incorpora-

so called

substituted

of choline

has been

The

The uptake

ferricyanide

in n e u r o b l a s < o m a

of choline

ones.

effect

choline

component,

in the

Substitution

an inhibitory

with

the

ions were

sucrose.

ting and d i f f e r e n t i a t i n g

choline

was higher

if sodium

line derived

investigated.

in p r o l i f e r a t i n g

or with

sodium produced line.

choline

than

inhibited

or lithium

ture

tumor has been

of 14C labelled

rentiating

in a cloned cell

1972;

in several

and Smyth, 1965;

Martin,

components

Yamamura

1969; 1968,

(March-

and Snyder,

3~

Pharmaco/ogical Research Communications, Voi. 5, No. 4, 1973

1972; that

Whittaker,

1972;

a carrier mediated

nedy,

1969;

lability

of ions

Haga and Noda,

in the

1973)

It has been cholinergic

(Hanin et al., of a suitable of mouse

model.

Under

tion of the m e c h a n i s m

in vitro

Neuroblastoma ding

choline

cell

metabolism

general

metabolism

thesis.

In the first

simple

situation

the perspective

by which

cultures : choline

morphological difference

as well

concerning

tic e q u i p m e n t

neurotransmitters. gical

and b i o c h e m i c a l

from the

et al.,

drastically

(Seeds

consumption

is increased

an investiga-

neuroblastoma

considered

clones

present

concerns

and

; the main

of the enzymaof

the m o r p h o l o -

serum w i t h d r a w a l

morphology

et al., 1970)

syn-

various

and d e g r a d a t i o n

a p p e a r after

for

neosynthesis.

or the absence

(Nissen et al.,

only

the most

characteristics

1970; H e r m e t e t

regar-

for a c e t y l c h o l i n e

In fact c e l l u l a r

of a c e t y l c h o l i n e s t e r a s e

cultures

for the

is used by the cells

characteristic which

choice

two p o s s i b i l i t i e s

neuroblastoma

events

i n c u b a t i n g medium~

activities

offer

for the synthesis

Another

of the

started.

as b i o c h e m i c a l

necessary

model

of a c e t y l c h o l i n e

the p r e s e n c e

study

that cloned

enters

step of our work we

It is well known that

1968;

by the

of the neuron

or it is used as well

in the absence

avai-

difficulties

a useful

choline

has been

the

can be avoided

metabolism

and Ken-

phenomenon.

technical

can represent

neuroblastoma

cultivated

several

shown

on the

(Martin~

in the past that

out

some of which

1970)

study of the c h o l i n e r g i c

cells

in this

is involved

Diamond

dependent

surroundings

cellular

system presents

It has been

1968;

strictly

1972),

pointed

1973).

(Potter,

process

et al.,

Green

and Noda,

Haga

changes

1972)

as well

; the O as the

catechol-O-methyl-

2

Pharmacological Research Communications, VoL 5, No, 4, 1873 transferase sidered 1970).

et

(Blume

as r e v e a l i n g The terms

in the context

a~.,

1970).

a cellular

with or without

fetal

phenomena

differentiation

proliferation

as i n d i c a t i n g

These

399 have been

#Seeds et aZ.,

and d i f f e r e n t i a t i o n

cultures

con-

will be used

which have been

incubated

calf serum r e s p e c t i v e l y .

METHODS. Clone N 18 which has been remarkable

acetylcholinesterase

vity and a barely (Amano et al., of clone

Falcon Eagle

1972).

N 18

tissue medium

atmosphere growth.

detectable

but no choline

findings

inactive

(Gibco,

flasks USA)

at

stage the

the flask and form a u n i f o r m

(6 cm ~) c o n t a i n i n g

zed. dium.

Krebs-Ringer Each

Petri

of 14C methyl Amersham, cubation

England)

was

chloride

The dishes

2 ml c o n c e n t r a t e d

were

formic

phase

Petri dishes

the cells were

activity

was d e t e c t a b l e

then dried After

utilime-

and 0.5

three

times

in-

with

in the third

and the cells checking

~Ci

60 mCi/mmole;

37oC. At the end of the

were r a p i d l y w a s h e d

acid.

as soon as

5 ml of the m e d i u m

at

of

surface.

used as i n c u b a t i n g

(specific

incubated

the Petri dishes

% Air

to the b o t t o m of

m e d i u m and,

again,

in

modified

a 5 % C02-95

Falcon

pH 7.2 was

NaCI 0.14 M. No r a d i o a c t i v i t y washing.

into

dish c o n t a i n i n g

choline

been c u l t i v a t e d

layer on all the plastic

was a t t a i n e d

phosphate

has

are a t t a c h e d

5 ml of D u l b e c c o

phase

activity

up to the s t a t i o n a r y

The cells were then t r a n s f e r r e d

acti-

ones.

37°C under

cells

a

classification

(75 cm 2) in Dulbecco

100 % humidity,

At this

the s t a t i o n a r y

allow the

study has

acety!ase

hydroxylase

(a gift of M. Nirenberg)

culture

and

in the present

tyrosine

The~e

N 18 among the

Clone

used

dissolved

in

under m i c r o s c o p e

Pharmacological Research Communications, Vol. 5, No. 4, 1973

400

the a b s e n c e was

of ce]!

transferred

omn~fluor then

debris,

~nto

sc~nt~]!at~on

(New E n g l a n d

counted

in an

SL 30.

Student's

gramma

602.

(Lowry

et

L• ' u _r ! e a ~. ,

t test was were

al~.quot of form~_c acid

counting

USA)

Intertechn~que

Proteins

a!. ,

a certain

calculated

assayed

an~. IO r~.! of

.. added . .Th~

were

Liquid

vials

vials

Sc{nt:[llation

with

Counter

an O ! { v e t t i

follow.ing the

were

Pro-

Lowry's

method

: 1 9 5 1 ) .

R E S U L T <' L.)

•

Choline tion

and

enters when

uptake

function

in d i f f e r e n t i a t i o n

the cells

following

a saturation

view of the tiated

as

cells

plateau

changes showed

which

of time

.is shown a straight

for cells

~n F~.g• 1• 14C c h o l i n e l~ne

up to about

starts

I:o develop•

follow

serum withdrawal,

a markedly

h~gher

in p r o l i f e r a -

uptake

As

2 hours

exnected,

in

d~.fferen-

of chol~ne.

CpM/pg Prot xlO 2 Fig, :1. - C h o l ~ n e u p t a k e as f u n c t i o n of t i m e in prol i f e r a t i n z and d i f f e r e n t i a t i n g cells.

D

,

©

3

O

/

o e -/

6

1 30

90

Min

Each point is the mean value of at least six Petri dishes• E x p e r i m e n t a l c o n d i t i o n s as in T a b l e 1.

Pharmacolog/cal Research Communications, Vol. 5, No. 4, 1973 Substitution cesium,

of s o d i u m

l i t h i u m or s u c r o s e

the

uptake

and

differentiating

potassium

of

was

choline

ions

produced

total

cells

a definite

same

with

inhibition

V a l u e s are e x p r e s s e d dishes.

was

(Table

as m e a n s

Proliferation

Incubation time

90 min

Control

210 ±14 A

Cesium

122

±17

Control

210

±14 C

Lithium

147

±12.4

observed

Control

237

±14 E

157

±11.1

Potassium

Control

210 ±14 G

Sucrose

129

when

1).

±S.E.M.

in

of 5 Petri

Differentiation

P

60 rain 208

±12

129

,7

208

,12

148

±4

0.005

0.005

on

in b o t h p r o l i f e r a t i n g

effect

sodium

medium with

1-. E f f e c t of ionic s u b s t i t u t i o n on c h o l i n e u p t a k e p r o l i f e r a t i n g and d i f f e r e n t i a t i n g cells.

Table

-

incubating

radioactivity

; the

substituted

in the

401

P

90 min

P

381 ±5 B 0.025

0.025

270

±4.5

381

,5 D

312 ,5

0.025

0.05

373 ±10 F 0.005

294

,13

0.005

381 ±5 H

±7.5

0.005

250 ±5

0.005

NaCI 138 mM was s u b s t i t u t e d by CsCi, LiCI or s u c r o s e in e q u i m o lar c o n c e n t r a t i o n s . KCI 5.5 mM was s u b s t i t u t e d w i t h e q u i m o l a r NaCI. I n c u b a t i o n was p e r f o r m e d in 5 ml K r e b s - R i n g e r p h o s p h a t e at pH 7.2 c o n t a i n i n g 10 % fetal calf s e r u m in the case of cells in p r o l i f e r a t i o n . P r o b a b i l i t y of s t a t i s t i c a l s i g n i f i c a n c e was c a l c u l a t e d w i t h S t u d e n t ' s t test (two tail). A vs B, C vs D, E vs F~ G vs H = P < 0.001.

Table

1 shows

Petri d i s h e s is o b s e r v e d

a statistical

analyzed

in p a r a l l e l .

i~ c o m p a r i n g

ferentiating

cells.

Petri

which were

dishes

analysis

This

the

High

controls

difference

statistical

on s e p a r a t e difference

of p r o l i f e r a t i n g

was

incubated with

performed

still

valid

substituted

and dif-

a m o n g the ions.

402

Pharmacological Research Communications, Vol. 5, No. 4, 1973

Table

2 - E f f e c t of o u a b a i n proliferation. Values

are

on

choline

expressed

as

uptaZe

means ,

Ouabain

cpm/pg

,j

protein

P

n

278

±5

5

260

±5

5

Control -4 10 M

258

±8

10

204

±3

15

278

,5

5

216

±3

5

3 - E f f e c t of o u a b a i n differentiation. Values

Ouabain

D hrs.

Control -4 10 M

24

Control -4 10 M

48

Control -4 10 M

72

Control -4 10 M

120

are

cpm/~g

on

expressed prot.

n

30q

±11

12

347

±23

12

445

±19

12

441

±22

12

886

±56

11

750

±51

13

878

±23

3

571

±53

3

choline as

means P1

IF;

|

0.05

0.025

0.005

P represents a two tail probability. Conditions as in T a b l e 1. I n c u b a t i o n t i m e 90 min.

Table

cells

±S.E.V.

Control -5 10 M

Control -3 10 M

in

of

uptake

in

incubation

celIs

in

±S.E.M. P2

P3

P4

n.s. 0.005 n.s.

0.0005

0.0025

0.0005

0.005

0.025 n.s.

0.0005

D hrs., hours after serum withdrawal - Pt, p r o b a b i l i t y (two t a i l ) of s t a t i s t i c a l significance between control Petri dishes and o u a b a i n t r e a t e d - P2, P3, P4, p r o b a b i l i t y (two t a i l ) of statistical difference a m o n g c o n t r o l P e t r i d i s h e s at d i f f e r e n t t i m e s of d i f f e r e n t i a t i o n - n.s.~ not significant - n, n u m b e r of Petri dishes. Conditions of i n c u b a t i o n as in T a b l e 1. P2 vs c o n t r o l 24 hrs. - P3 vs c o n t r o l 48 hrs - P4 vs c o n t r o l 72 hrs.

Pharmacological Research Commun/cations, Vot. 5, No. 4, 1973 Ouabain and

10 -3 M)

ration ved

(Sigma Co. , USA) inhibited

(Table

in cells

withdrawal

P2,

P3,

for longer

statistical

time

significance

in p r o l i f e -

(10 -4 M) was obser-

72 hrs.

after

from Table

showed

10 4

serum

3 cells

a h i g h e r uptake

is shown

in the

kept

of

columns

P4.

ferentiation (Table

4).

was

affected

a drastic

Finally

bition of the uptake

4

(4°C)

producing

nal concentration)

Table

starting

(10 -5,

in cells

of ouabain

3). As can be observed

Low t e m p e r a t u r e

tion

concentrations

of choline

same effect

in d i f f e r e n t i a t i o n

(Table

the

the uptake

2). The

in d i f f e r e n t i a t i o n choline,

at different

403

when

decrease

potassium

added

was

both p r o l i f e r a t i o n

to the

in choline

ferricyanide

incubating

again o b s e r v e d

and dif-

incopora(10 -3 M fi-

medium,

an inhi-

(Table 4).

Effect of cyanide and low t e m p e r a t u r e on choline take in p r o l i f e r a t i n g and d i f f e r e n t i a t i n g cells. Values

are e x p r e s s e d

as means

±S.E.M.

Pro I i ferat ion cpm/ug

prof.

n =

up-

Differentiation P

cpm/ug

:

prot. - -

n

P

,

CYANIDE Control -3 10 M

270 ±6

9

239 ±9

10

328 0.005

~10

5

251 ±10

5

374 ,10

5

0.0005

TEMPERATURE Control

277

4°C

±14

5

11 ±1 ,,

,,,

,

5 r,,

0.0005 ,

.

.

.

18 ±4 .

_ _

5

0.0005

__ --

j,

m ,

n, number of Petri dishes ; P, p r o b a b i l i t y of s t a t i s t i c a l difference (two tail test). C o n d i t i o n s of i n c u b a t i o n as in Table I. Incubation time 90 rain.

DISCUSSION Cells

in d i f f e r e n t i a t i o n

uptake of choline

than those

clearly

showed

in p r o l i f e r a t i o n

a more p r o n o u n c e d (Fig.l).

From

404

Pharmacologica/ Research Comrnunicadons, Vol. 5, No. 4, 1973

Table

3 it a p p e a r s

three

folds

uptake

that

increasing

this the

of d i f f e r e n t i a t e d

seen

from the

Such

effect

occurs

values

may

after

serum

of c h o l i n e

membranes

; however

sibility

of a c h a n g e

the

from

P2 at the to the

velocity

and

the

significant right

!ipids,

as

can

be

3.

surface

increase

basic

Such

of T a b l e

in m e m b r a n e

to the

kept

up to a b o u t

wJthdrawa!.

extreme

and/or

to be

in the

serum

increase

containing it has

increased

Js highly

withdrawal

synthesis

affect

time

cells

of

be due

difference

which

in the

components

in c o n s i d e r a t i o n

structure

of the

membrane

mechanism

of the t r a n s p o r t

of the

the

pos-

which

may

of cho-

line. In o r d e r sodium

or p o t a s s i u m

medium.

a decrease

that

Li + and

sodium-potassium It is then

tained

was

of the

possibilities

crose

been

of

sodium

pump

the

: the

lack

one

of the

sodium

As

cation

produced and

1964;

the

et oZ.,

decrease direct

ob-

inhibi-

substitutes. observed to take or the

substitu-

inhibition

is p r o d u c e d of the

Na + pump,

a decrease

differentia'ring

with

into a c c o u n t

in v i e w

of the

also

Beaug6

or to the

which

expected,

sucrose,

on the

whether

ATPase

or

action

again

functioning

lithium

in the

inhibitory

by the

has

substituted

been

in u p t a k e

activated

data).

in p r o l i f e r a t i n g

state

mechanisms,

it has

of Na + ions

decrease

for the by

to

such

; however

and Ager,

produced

sucrose

(unpublished

of p o t a s s i u m

a direct

difficult lack

cesium,

uptake

(~ittam

on

entirely

with

in c h o l i n e

extracellular

ce of K + ions

take

have

Cs + have

sodium

of Na + with

Na + - K +

ions

due to the

Concerning tion

information

pump

1973).

tion

some

Substitution

produces shown

to g a t h e r

cells.

two

of by

su-

importan-

substitution

of c h o l i n e Increasing

up-

Pharmacologica/ Research Communications, Vo/. 5, No. 4, 1973 sodium concentration

in the medium did not influence the upta-

ke. The data demonstrate ke in neurob!astoma

405

the strict dependence of choline upta-

cells

in the incubating medium

to the presence

of Na + and K + ions

; dependence which

is again shown by

the inhibitory action of ouabain and by the necessity of potassium ion.

It is also possible

choline across

clone N18 cell membrane

mechanism since cyanide lowered

of

is an energy-dependent

and low temperature

have considerably

the uptake of choline.

Furthermore ne transport by an

to ~nfer that the transport

the presence of carriers

in neuroblastoma

for choli-

cultures has been recently

analysis of the kinetics

as well as in a cholinergic

necessary

of choline uptake

clone

found

in clone N18

(in preparation).

REFERENCES. Adamic, ~ , (1970): Biochim. Biophys. Acta 169, 113. Adamic, S., (1972): Biochem. Pharmacol. 21, 2---~25. Amano, T. , Richelson, E. and Nirenberg, M.W. , (1972) : Proc. Natl. Acad. Sci. USA 69, 258. Augusti-Tocco, G. and Sato, G., (1969): Proc. Natl. Acad. Sci. USA 64, 311. Beaug~, L.A., Medici, A. and Sjodin, R.A., (1973): J. Physiol. 228 , 1. Blume, A., Gilbert, F., Wilson, S., Farber, J., Rosenberg, R. and Nirenberg, M.W., (1970): Proc. Natl. Acad. Sci. USA 67 , 786. Diamond, I. and Kennedy, E.P., (1969): J. Biol. Chem. 244, 3258. Diamond, I. and Milfay, D., (1972): J. Neurochem. 19, 1899. Green, A.R., Boullin, D.J., Massarelli, R. and Hanin, I., (1972): Life Sci. 11, 1049. Haga, T. and Noda, H., (1-973): Biochim. Biophys. Acta 291, 504. Hanin, I., Massarelli, R. and Costa, E., (1970): Drugs and cholinergic mechanisms in the central nervous system, E.Heilbronn and A. Winter, Eds., Res. Inst. Natl. Def., Stockholm. Hanin, I., Massarelli, R. and Costa, E., (1972): J. Pharmacol. Exper. Therap. 181, 10. Hermetet, J.C., Ciesie--~ki-Treska, J. and Handel, P., (1972): J. Histochem. Cytochem. 20, 137. Hodgkin, A.L. and Martin, K., ~-f965): J. Physiol. 179, 26. Lowry, O.H., Rosebrough~ M.J., Farr, A.L. and Randall, R.J., (1951): J. Biol. Chem. 193~ 265.

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