Interactions between ionophores, Mg2+ and Ca2+ on acetylcholine formation and release in brain slices

Pharmacological Research Communications, Vol. 13, No. 2, 1981 INTI,:RACTIO[,IS ]]ETWEEN IONOPHOHES, FOR[,IATION AND R E L E A S E

P. M a n t o v a n i Department Morgagni

Mg 2+ AND Ca 2+ O1': ACETYLC, HOLINE

IN B]~AIN SLICES

and G. Pepeu

of P h a r m a c o l o g y ,

65,

175

50134

University

of Florence,

Viale

Florence, I t a l y

SU ~,~,iAR Y The effect the i n c u b a t i o n ACh output BrX-B37A

of different medium

with

(Bromolasolacid) were

solution

put was m e a s u r e d BrX-537A effect

enhanced

synthesis

in

slices and on their

of the t w o ~ i o n o p h o r e s

and A 23187 was studied. from rat cerebral

cortex,

incubated

ACh c o n t e n t

and out-

by bioassay. ACh output

also in C a 2 + - f r e e The a d d i t i o n

medium

and total ACh

formation.

effect

to the i n c u b a t i o n

of B r X - ~ 3 V A

on ACh output.

that C a 2+ and Mg 2+ c o n c e n t r a t i o n

primarily

The

m e d i u m and in C a 2 + - e n r l c h e d

o f Mg 2+ (9.3 ~1)

the s t l m u l a t o r y

It is concluded incubation

the addition

prepared

stimulated

occurred

in brain

c o n t a i n i n g physostigmine.

(5.0 ~,I) medium. medium

on ACh level

and without

The slices in Krebs

Ca 2+ and Ng 2+ c o n c e n t r a t i o n s

modulate

and exert only an indirect

ACh release, influence

in the

cor~tent and

on BrX-53?A.

INTROOUCT!ON Acetylcholine about

by c a l c i u m

(ACh)

fluid

thereby d e p r e s s e s

from nerve

entrs, (Katz and Miledi,

Mg 2+, a p r e d o m i n a n t l y extracellular

release

Intracellular

competes

ACh output

0031-6989/8 I/'020175-10/$02.00/0

endings

1965;

cation,

is br~uF~ht

Miledl,

1973).

when present i n

with Ca 2+ for active from motor nerve

sites

terminal

the

and (Del

0 1981 The itai,an Pharmacolog,cal Society

PharmacologicalResearch Communications, I/ol. 13, No. 2, 1981

1 76

C a s t i l l o and Engback, sympathetic

ganglia

Substitution

1954, Hubbard,

(Hurter and Kostlal,

1954).

of Ca 2+ with Mg 2+ strongly

from the frog spinal cord (Nistrl, s ynthesis and release stimulat e d the isolated

et al., 1968) and from

1976).

Mg 2+ inhibits ACh

by high K + c o n c e n t r a t i o n

ileum (Gerhards, et al., 1964)

(Molenaar and Polak,

reduced ACh release

and in b r a i n slices

1970).

E x t r a c e l l u l a r Mg 2+ seems t h e r e f o r e to depress A C h in all e x p e r i m e n t a l an

in

conditions

so far investigated.

Increase in Mg 2+ c o n c e n t r a t i o n

output

However,

in the incubation medium

p o t e n t i a t e s the s t l m u l a t o r y effect o f the ionophore B r X - 5 3 7 A (Bromolasolacid) et al., 1978),

on A C h output

from brain slices

(Casamenti,

In the present paper this e f f e c t of. Mg 2+ is

i nvestigated by studying

the interactions

between B r X - 5 3 7 A ,

Ca 2+ and Mg 2+ on ACh formation and release.

M A T E H I A L S A_ND [~ETHODS Slices were prepared

from the cerebral cortex of adult

male Wistar rats killed by decapitation. incubated at 37 ° C in Krebs sol u t i o n tion (raM):

The slices were

of the followinF~ composi-

NaCI 113, KCI 4.7, CaCI 2 2.5, KH2PO 4 1.2, MgSO 4 1.2,

NaHCO 3 25, glucose 11.5,

p h y s o s t l g m l n e sulphate

1.54 uM.

The

s o l u t i o n was gassed with 5% CO 2 in 0 2 . In some experiments modified Krebs solutions were used: free solution was obtained by omission of CaCl2;

Ca 2+-

Mg 2+ was raised

to 9.3 mM and Ca 2+ to 5 mM by a d d i t i o n of M g S O 4 or CaCl 2 respectively.

In the latter case Ns~{CO 3 was also s u b s t i t u t e d

with NaCl

in order to avoid Ca precipitation. ACh ~as extracted Beani et al.

from the slices a c c o r d l n K

to the method o f

(1963), either at the end of 60 mln p r e i n c u b a t l o n

(O time) or at the end of the following 60 rain incubation.

ACh

Pharmacological Research Communications, VoL 13, No. 2, 1981 o f the e x t r a c t s

was measured

~ig ileum a c c o r d i n g After

60 mln

and ACh o u t p u t

177

by b i o a s s a y

to Beanl

et al.

preincubation,

on the isolated

~ulnea-

(1978).

fresh medium was s u b s t i t u t e d

in the i n c u b a t i o n

m e d i u m was measured

e i t h e r every

I0 mln or at the end of 60 rain incubation. Sodium-dependent by the method

affinity

of S i m o n et al.

from the incubated 130 Ci/mmol

hiF~

brain slices.

containing

(0.25 mg of protein)

over M i l l i p o r e

transferred

samples

filters

of 20 mm Jig.

ethylether

Methyl

and

were c o u n t e d

filters vials

Instagel

incubated

Manifold

expressed

method.

as pmol of choline

with

were a l l o w e d

was added

of choline in ~ O u b n o f f ~oured

a suction

to dry and were

to vfnich ethylenc-lycol-mono-

(Packard)

in a Packard

standard

chloride

of s y n a p t o s o m e s

concentration

were added

Triearb

(mod 577)

tion s p e c t r o m e t e r with a counting, e f f i c i e n c y by the e x t e r n a l

choline

The content of the tubes was

on a M i l l i p o r e The

obtained

- Amersham,

The tubes were

to s c i n t i l l a t o r (Merck)

3H

Centre

to o b t a i n a final

s h a k e r at 30 ° C for 4 rain.

was measured

in s y n a p t o s o m e s

a resusper:ded a l i o u o t

in the m e d i u m o f 0.04 uM.

pressure

(1976)

from the R a d i o c h e m l c a l

to the tubes

c h o l i n e uptake

and

the

scintilla-

of 35% d e t e r m i n e d

The rates of c h o l i n e uptake

per 4 min i n c u b a t i o n

v,ere

time per mF, of

prote in. The method

protein

content o f the s y n a p t o s o m e s

of Lowry et al.

Concentrated were made

attained

solutions

(I - I0 mg/ml) and w e r e

The h i ~ h e s t c o n c e n t r a t i o n

in the i n c u b a t i o n m e d i u m was

no e f f e c t on ACh o u t p u t and c o n t e n t to all controls.

by the

(1951).

in d i m e t h y l s u l p h o x i d e

as necessary,

was m e a s u r e d

of B r X - 5 3 7 A and A 23187

diluted

500 to 200 folds

of d i m e t h y l s u l p h o x i d e

25 m~4 and while

this had

it was n e v e r t h e l e s s

added

178

Pharmacological Research Communications, VoL 13, No. 2, 1981

EE SULTS Fig 1 shows every

the e f f e c t

of B r X - 5 3 7 A

10 rain in the presence

i n c u b a t i o n medium,

Of two Mg 2+ c o n c e n t r a t i o n s

increase

6 times

larger

It can be seen that the m a x i m u m

in ACh output e l i c i t e d

in presence

by B r X - 5 3 7 A

a s e c o n d ionophore,

to the i n c u b a t i o n medium at the c o n c e n t r a t i o n

increase

1978),

caused a 2 8 0 %

increase

A 23187,

was 6 3 2 % with Mg 2+ 9.3 raM.

3000

WITH

Mg 2.9.3 mM

LtJ

Z

2000

g ID a. ID

1000

O U <[

Mg ~" 1.2 mM 200 L

,J

0.15

0 45

....

0.9

CONCENTRATIONS

Fig.

I

1.8 OF

3.6

BrX~S37A

(pM)

added

of 58 uM (Casamenti

in 4 e x p e r i m e n t s

Brx-537A DOSE.EFFECT RELATIONSHIP D I F F E R E N T Mg 2÷ C O N C E N T R A T I O N S

U) < UJ nO

1.8 uM was

of 9.3 than in that of 1.2 mM Mg 2+.

Under the l a t t e r c o n d i t i o n s

et al.,

in the

With Mg 2+ 9.3 ~ff4 the output after B r X - 5 3 7 A

was larger than with 1.2 n~4. percent

on ACh output m e a s u r e d

v~ile the

Pharmacological Research Communications, Vol. 13, No. 2, 1981

179

However, by e x p r e s s i n g t h e effect of the ionophores on ACh output

as percenb

ences

caused

increase

o v e r the basal

b v t h e two Mg 2+ c o n c e n t r a t i o n s

ent if h i g h Mg 2+ also d e p r e s s e s son b e t w e e n therefore slices

the absolute

be made'

o f ACh

differ-

would o n l y b e

the basal ACh output,

values, r e p o r t e d

Table

at the b e g i n n i n g

the amount

output, t h e

1 also

shov~

appar-

A comparl-

in table I, should

ACh content

in brain

and at the end of 60 mln incubation,

total ACh formed d u r i n g k i n the presence o f d i f f e r e n t Mg 2+ and C a 2+

incubation

r e l e a s e d a n d the

concentrations. The changes

content after

in i o n

i

of theslices

concentrations

at

the beginning

60 mln preincubation).

in C a 2 + - e n r i c h e d solution.

affected

ACh

of the incuba~ton

ACh content

"(5.O m M ) a n d

Increasing

firstly

was much lower

in C a 2 ÷ - f r e e

Mg 2+ c o n c e n t r a t i o n

(i.e. both

than in n o r m a l Krebs

caused

a slight

increase

in ACh content. The r e l a t i v e l y brain slices

small

in normal

basal A C h

Krebs

output

solution

from the u n s t i m u l a t e d

was decre~u;ed by the r e m o v a l

of Ca 2+ and b y the increase i n Mg 2+. On the contrary it v~s \ s t r o n g l y enhanced by d o u b l i n g Ca 2+ c o n c e n t r a t i o n and by the addition

of BrX-537A.

Little incubation both

or no ACh was formed in normal

the a b s e n c e

markedly however

or M g 2 + - e n r i c h e d

ACh

formation.

of the ACh

in the slices

was much

formed lower

was

ACh

of BrX-537A

formation.

on ACh o u t p u t

In h i g h

always

was twice,as

solution.

In the latter released

60 min Conversely

conditions

and ACh c o n t e n t Krebs.

in the i n c u b a t i o n

stimulated

M~,2+ Krebs

during

of its c o n c e n t r a t i o n

than in normal

h~latever the ion c o n c e n t r a t i o n the a d d i t i o n

Krebs

of Ca 2+ and the doubling

stimulated most

by brain slices

solution

ACh release the e f f e c t

large as in.normal

Krebs.

medium, and total of BrX-537A However

an

I: E F F E C T

a

a

d

b,c v e r s u s

d versus

e,f v e r s u s

edlfferent

1 with

P
P<0.01

P
0

0 1.8

1.8

a

g versus h versus

b

e versus

P<0.01

versus

p,q v e r s u s

P
r

n versus

i versus

(6) (4)

(4)

(12)

(4)

P <0.05

P<0.02

2.72+0.28 i 5.06+0.611

1.03+O.09 h

0.31+0.03g

3.21+-0.38 f

O. 25~--0.o15d (15) 3.35+0.28 e (6)

0 1.8 3.6

1 . 6 7 + 0 . 3 b (i0) i.O7+0.13 c (6)

0.43+0.21 a (21)

ACh o u t p u t d u r i n g 60 min

S

o

m

b,i

A/~D 0 U T P U T , E X P R E S S E D

1.8 3.6

0

BrX-537A uN

0N ACh C C N T E N T

of e x p e r i m e n t s .

2.79+0.120 (4)

number

from

In p a r e n t h e s i s

Mg2+ 1.2 mM Ca 2+ 5.0

(4)

5,06+0.95~

Mg2+ 1.2 mM

mM

(8)

Ca 2+ 2.5 mM

Ca 2+ 0

12.854"0.76

llg 2+ 9.3 ~

"

(12)

"

9 62+0 76 1

ACh c o n t e n t at 0 time

OF B r X - 5 3 7 A

C a 2+ 2.5 mM

;4g 2+ 1.2 mM

Conditions

TABLE

+ S.E.,IN

(4)

P<0.05

P<0.05

P<0.05

P<0.01

t lu v e r s u s

SLICES

m

P
.O1

P
5.96 6.94

7.71

2.77

4.93

5.84

6.92 1.16

0.87

Total ACh formed in 60 m i n

BRAIN

r versus

6 . 0 3 + 0 . 8 t (4) 4.67+0.35 u (4)

1 1 . 7 4 ±I.06 s

7.52+-0.82 r (4)

1 4 . 5 7 + 1 . 5 7 q (4)

11.34+0.92 ° (8) 15.34+1.28 p (4)

1 4 . 8 7 + 0 . 6 4 n (4) 9.71+0.5 (4)

10.06±1.01 m (8)

ACh c o n t e n t a f t e r 60 rain

AS ug/g

o0

CO

c%

cb

c~

0

Pharmacological Research Communications, Vol. 13. No. 2. 1981

increase Krebs

in the synthesis

was

Even

exerted

in C a 2 + - f r e e

(HC-3)

t i o n did not e l i m i n a t e uM) on ACh o u t p u t . was only

a difference

Drain

was

obtained 1980).

effect

percent

in s y n a p t o s o m e s DrX-537A

solu-

of BrX-537A over

protein

prepared

choline

At

solution hich

Drool/4 m i n / m g

to

(i.8

the basal (n=15),

significant.

investiKated.

Krebs

able

ME 2+ K r e b s

increase

of B r X - 5 3 7 A on hi qh affinity

in normal

I.I + 0.07

to high

from 1572 +- 200 to II12 +- 153%

s l i c e s was also

incubation

~ras still

formation.

10 -2 added

not s t a t l s t i c ~ l l y

The e f f e c t

BrX-537A

the s t i m u l a t o r y

reduced

A similar

Krebs solution.

and ACh

The maximum

in normal

dose o f BrX-537A.

Krebs s o l u t i o n

ACh output

Hemicholinium-3

output

t h e largest

in C a 2 + - e n r i c h e d

stimulate markedly

the

of ACh over that o c c u r r i n E

was only s e e n with

effect

181

in

the end of 60 rain

affinity- choline

(n=4):,

from

uptake

A

s i m i l a r value

fresh slices

1.8 uM did not a f f e c t h i K n

untake was

(Pedata

et al.,

uotake

neither

affinity

in 1 . 2 nor in 9.3 mM i4~2+ i n c u b a t i o n m e d i u m .

D I SC US SION U n d e r restinK slices .incubated

conditions

i n normal

the additio~

Krebs

solution

increase

in A C h

output

A direct

effect

of the parent

ionophore

been excluded

by R i c h t e r

transferase enhanced uptake

has

ACh c o n t e n t

%.Jhose rate

content Wen solution

in the slices

is i n v e r s e l y

(Roskowski, brain

indirectly

1978;

slices

the present

a marked

activatinF, also ACh synthesis. X-537A

on choline

(1977)

~rX-5~7A

and hip.h a f f i n i t y

related

1978)

of BrX-537A

experiments

was

acetvlelso

choline

to s y n a p t o s o m a l

?~esleP et al.,

output

to brain

brouKht a b o u t

ACh

:.:as n o t stimulated.

~'iere i n c u b a t e d in ~4g2+ enrie~ed

the e f f e c t on ACh

tions used i n

of B r X - 5 3 7 A

at both

ootentinted

Krebs concentrabut only at

Pharmacological Research Communications, Vol. 13, No. 2, 1981

182 the largest

concentration

enhanced.

ACh

incubation

in Mg 2+ enriched

medlum

content

the effect

and t h e r e f o r e

ACh store

bigger

release

was

a t the b e g i n n i n g

could have

of the

than in normal

acted u p o n a larger

release.

can be raised

ACh f o r m a t i o n a n d

formation

m e d i u m was larger

BrX-SB7A

stimulating

The q u e s t i o n

in the slices

on ACh

as to w h e t h e r B r X - 5 3 7 A

only

through

its

stimulates

ionophoric

properties

or by some other mechansims. Richter

(1977)

the analogue duced and

showed

X-B37A o n

a similar

Sokolowsky

(1978)

that in Ca2+~-free medium the effect

ACh r e l e a s e

observation

in Torpedo

reduced

The p o s s i b i l i t y

that

cellular

has been s u g g e s t e d

stores

We observed solution

still

endings,

unrelated

released ACh,

was a p p a r e n t l y

larger t h a n in normal (1966)also

lobster n e r v e

was

showed

larger

(Tucek,

activity

Ca2+-dependent.

is not

The s t l m u l a t o r y neurotransmltters

it has

agent

is reduced (Holz,

been claimed

increase

effect

1977;

transferring Ito et al.,

cations 1978;

output

Dettbarn in the sea w a t e r

acetyltransferase

1977; Ito et al.,

by d e p o l a r i z a t i o n (~asamentl

and Sokolovsky,

of several

m e d i u m added

and its a n a l o g u e

as an i o n o ~ h o r e

Michaelson

formation

in C a 2 + - f r e e

Richter,

that B r X - B B T A

neurotransmitter

solution.

synthesis

of B r X - 8 3 7 A on the release only

Krebs

from cut nerve

than in normal

I'9?7) that choline

1978).

in Ca2+-free

and their ACh

that ACh

in C a 2 + - f r e e

and it is known

and Currel,

a leakage

Krebs

on

Ca 2+ from intra-

incubated

activity,

of BrX-537A

and accumulation.

(Nordman

probably

to neuronal

and R o s e n b e r g

chelating

the effect

might release

that brain slices

and

In our e x p e r i m e n t s

but did not affect ACh f o r m a t i o n BrX-537A

was not re-

was made by M i c h a e l s o n

synaptosomes.

the lack of Ca 2+ only s l i g h t l y ACh output

from brain slices

of

1978)

and

X - 5 3 7 A could as well as by

et al.,

1978).

with

1978;

a

Pharmacological Research Communications, VoL 13, No. 2, 1981

Finally, reduced ACh

d o u b l i n g C a 2+ c o n c e n t r a t i o n

level in the

£anglion

increased

ACh output,

by H u r t e r and Kostial

and ACh synthesis.

In C a 2 + - e n r i p h e d increase amount

in the I'.rebs solution

slices and m a r k e d l y

as shovn~ in the per'fused cervical (I~54),

183

the already

of ACh

at the expense

Krebs s o l u t i o n

released

in normal

of ACh content,

Krebs.

the same

This occurred

partly

partly by stimulatin}~ ACh s y n t h e s i s

an additive

effect of B r X - 5 3 7 A

and h i g h Ca 2+

on ACh output.

In c o n c l u s i o n ,

these

results d e m o n s t r a t e

brain slices Ca 2+ and Mg 2+ c o n c e n t r a t i o n s influence

was still able to

larRe ACh output by p r a c t i c a l l y

This finding s u g g e s t s concentration

BrX-537A

on B r X - 5 3 7 A

that in resting

exert an i n d i r e c t

a c t i o n by m o d u l a t i n g ACh

release, content

and synthesis.

ACKNOh;LEI)GENENTS This work was C.I!.R.

s u p p o r t e d b y grant n~ 7 8 , 0 2 2 2 6 . 0 4

BrX-537A.was

a generous

gift of Roche

from the

and A 23187

of

Ely Lilly Co.

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