Neosurugatoxin blocks nicotinic acetylcholine receptors in the brain

Neurochem. Int. Vol. 7, No. 2, pp. 38%396, 1985

0197-0186/85 $3.00 + 0.00 Pergamon Press Ltd

Printed in Great Britain

RAPID IMPORTANT PAPER NEOSURUGATOXIN Catherine

BLOCKS

Rapier,

Department

NICOTINIC

Roger

ACETYLCHOLINE

Harrison,

of Biochemistry,

George

University

RECEPTORS

G. Lunt

IN THE BRAIN

& Susan W o n n a c o t t

of Bath,

Bath BA2

7AY,

U.K.

(Received 3 December 1984; accepted 4 February 1985) ABSTRACT Neosurugatoxin, a neurotoxin isolated from the Japanese ivory mollusc (Babylonia japonica) is a nicotinic antagonist with a s p e c i f i c i t y towards ganglionic nicotinic receptors. At low concentration (5 x 10-'M) neosurugatoxin inhibited the release of ['H]dopamine evoked by l,l-dimethyl-4-phenylpiperazinium (DMPP) [rom rat striatal nerve terminals, without affecting the response to K---depolarisation. In contrast, sbungarotoxin did not a n t a g o n i s e the action of DMPP. Neosurugatoxin also i n h i b i t e d ['H] nicotine b i n d i n g to rat brain m e m b r a n e s but had no effect on [~zsI]sbungarotoxin b i n d i n g to the same tissue preparation. These results support the v i e w that functional nicotinic receptors in the CNS resemble g a n g l i o n i c n i c o t i n i c receptors. N e o s u r u g a t o x i n has c o n s i d e r a b l e potential as a useful probe for such receptors in the brain.

There

is

an

receptors

increasing

in

acceptance

vertebrate

understanding

the

CNS

particular

to the w e l l - c h a r a c t e r i s e d hampered

by

lack

sbungarotoxin,

of

that

Raftery,

1982)

sbungarotoxin 1982;

population

many neuronal

functional synaptic terminals have

and

et al.,

1982)

nicotinic

assay

system

nicotinic (Mills

demonstrated

responses to for

The

junction

our

of

1984;

However,

antiserum

et al.,

raised

389

release 1985).

against

(Norman

et

to a

has been

antagonist

of

1982).

studies

in w h i c h

[3H]dopamine

brain

selectively

an

&

There

between

its u s e f u l n e s s

& Dolly,

binding

of n i c o t i n i c

receptors

as

been

toxin,

receptor.

toxin may bind

receptors

Rapier

snake

in

relate

(Conti-Tronconi

crossreactivity

ineffectiveness

(see B a r n a r d

nicotinic

facilitation

an

its

they

has to some extent

probes.

nicotinic

that this

complement

& Wonnacott, that

peripheral

of

progress

and how

to our u n d e r s t a n d i n g

receptors.

in v i e w

acetylcholine

However,

receptor

immunological

proteins

sought

nicotinic

receptors

suitable

so much

binding

nicotinic

these

nicotinic

Wonnacott

have

of

ganglia.

to the study of the neuronal

including

particularly

presence

and at the n e u r o m u s c u l a r

applied

evidence,

of neuronal

questioned,

We

has been

of

of

contributed

in e l e c t r o p l a q u e s

al.,

functions

peripheral

has

the

autonomic

availability

receptors

is c o n s i d e r a b l e

of

and

the

by

developing

we m e a s u r e from

the

striatal

a

prenerve

In such a system we nicotinic

receptor

390

Rapid Important Paper

In each as

experiment

a control

3 perfusion

without

spontaneous

toxin

release

subtracted

from

chambers

and

throughout

the

were operated

the

second

the

perfusion

transmitter

as

release

the

in parallel:

test

period

was

profiles

one

system.

of

In

measured the

served

the

and

third

this

control

was

and

test

cortices,

and

systems.

Radioligand

binding

A crude reconstituted (w/v)

azide,

[3H]nicotine

buffer,

to give

binding

the presence

were

60

Bound

The

22°C,

in

radioligand

Microcentaur

Drugs

were of

9.4

CaCI,

as d e s c r i b e d

in 50 mM-HEPES

(2mM),

for

10mg/ml.

MgS04

60 min at

Bound r a d i o l i g a n d

(2mM).

22°C,

in

was separated

diluted

and

presence

separated

by

was

ten-fold

the

incubated

with

with

absence

assayed

by the

method

in 50 m M - p h o s p h a t e

of

2.5

x

(2

was w a s h e d

of

buffer,

1 nM-[12sI]abungarotoxin

centrifugation

the pellet

the

membrane

Non-specific

the conditions

i.i fmol/mg

respectively

membranes

for

10-SM-ebungarotoxin.

min

at

once with

10,000g;

MSE

1.5 ml ice-cold

saline.

under +

P2

incubated

radioligand.

subtracted; and

(4.8mM),

and 0.01%

approximately

five-fold

40nM-[3H]nicotine

to

was

centrifuge);

phosphate-buffered

addition

KCI

0.1mM-PMSF

essentially

was diluted

(0.5 ml) were

was

bench

binding

fraction

and aliquots at

of

was p e r f o r m e d

(ll8mM), with

rat brain

at 4°C.

(1977).

min

concentration

The tissue

NaCl

from

containing

of 10-~M-L-nicotine.

[z25I]abungarotoxin Schmidt

pH 7.4,

a protein

incubated

and absence

by filtration

prepared

buffer,

(1980).

containing

(0.25mi)

pH 7.4,

(P2) was

to this p r e p a r a t i o n

& Goldstein

pH 7.4,

Samples

fraction

in 50mM-phosphate

sodium

by Romano

assays

membrane

used

protein

(mean + S.E.M.

for

fraction

binding

specific

for

for

binding

[3Hlnicotine

3 membrane

10

(excess

min

prior

cold

was

and

to

the

ligand)

was

44.7 + 8.8 fmol/mg [~25I]ebungarotoxin

preparations).

RESULTS

Neosurugatoxin striatal

greatly

of the

ganglionic

peaks

of

(5 x i ~ ) ib)

(Fig.

nicotinic

released

only

control

21%

(Fig.

3

independent

affected. the

response

After to

and

agonist

15%

la).

DMPP

average

59 + 8%

DMPP

was

with

gradually

or

the

K+-evoked

restored

of

(83%

was

not

medium

without

after

20 min

from

low doses

elicit

sharp

neosurugatoxin 1 and 3 (Fig. peaks

DMPP-evoked

of duplicate

release

perfusion

peaks

corresponding

inhibition

la),

(28mM) with

was reduced:

of

[3H I dopamine

(Fig.

KCI

perfusing

(mean + S.E.M.

normal

of

of toxin

i~)

response

experiments). washing

(5 x After

respectively

The

was

release

In the absence

radioactivity.

5 x l~'M-neosurugatoxin from

the D M P P - e v o k e d

I).

for 20 min the D M P P - e v o k e d

represent

normal

diminished

synaptosomes

in the

release

by

determinations significantly neosurugatoxin washing,

109%

Rapid Important Paper purified

from

rat muscle

(Mills & Wonnacott,

inhibits

1984)

the

suggesting

nicotine-evoked

abungarotoxin

preparation

whereas

did

not

response

neurotoxin

from

isolated

called n e o s u r u g a t o x i n a

specific

compare

action

of

in

of

[3H]dopamine

ivory

However,

effect

mecamylamine

& Wonnacott,

Japanese

some

our

receptors.

pronounced

antagonist

(Mills

the

a

(Kosuge et al.,

antagonist

its

have

the ganglionic

the n i c o t i n e - e v o k e d

release

some degree of identity b e t w e e n p e r i p h e r a l

receptors and this group of central nicotinic contrast

391

1984).

mollusc

on

neuronal

nicotinic

system

(Babylonia

its

blocked

Recent reports

of a

japonica)

and

1984) which may be

receptors,

with

striatal

completely

1982; Hayashi et al.,

functional

in apparent

the

prompted

effect

us

to

in c o n v e n t i o n a l

b i n d i n g assays. MATERIALS

D,L-[N-methyl Radiochemicals (46

Ci/mmol)

U.K.)

'H ] nicotine

Ltd.

(73.7 Ci/mmol)

(Southampton,

were

purchased

~bungarotoxin

from

Hants.,

was

Ltd., Ltd.

in

of

(Gillingham,

Dorset,

Dorset,

the

rat

striata

final

were

suspension

prepared

as

protein

at 37°C

suspension were

(Rapier et al., Millipore

Krebs m e d i u m at a constant

glass

rate

The to

Chemical

Co.

described

['H]dopamine

Samples

filter

units)

fractions

of

successive the

and

in the perfusion with

modified

(i00~i) of KCI and the

was

Continuous achieved

by

allowing a 20 min

c o l l e c t i o n of consecutive using

an

LKB

Redirac

(counting e f f i c i e n c y

The Netherlands) 40%).

in a Packard

scintillation

0.2

fraction

r a d i o a c t i v i t y was c o u n t e d after addition of 3 ml of Aqualuma

Ad Schaesberg,

& of

(0.i M; 15 Ci/mmol)

perfused

d e p o l a r i s i n g pulses

pulses.

perfusate

Mills

(250~i) of the synaptosome

agonist DMPP were applied in the perfusion medium, between

by

concentration

(9ml/h).

After a 20 min w a s h o u t period,

B.V.

1985).

a

t r a n s f e r r e d to W h a t m a n GFF filters p o s i t i o n e d

(modified

collector;

diluted

(Poole, Dorset,

from A l d r i c h

having

The s y n a p t o s o m e s were incubated with

ml

Kosuge.

and

Drugs and toxins were serially diluted in p e r f u s i o n

for 4 min

interval

T.

and all other reagents were from BDH Chemicals

3mg/ml.

nicotinic

and

just prior to use.

from

(1984),

chambers

was

Bucks., U.K.)

1980).

dimethylsulphoxide

(DMPP)

NEN

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

Synaptosomes Wonnacott

U.K.)

U.K.)

m e d i u m or assay buffer Preparation

Sussex,

L - n i c o t i n e was from the Sigma Chemical Co.

l,l-dimethyl-4-phenylpiperazinium

(Poole,

(Amersham,

(Lewes,

(Wonnacott et al.,

0.12 ml

from

[*2SI]Na and 7 , 8 [ 3 H ] d o p a m i n e

n e o s u r u g a t o x i n was a gift from Professor

dissolved

3.0 ml with water. U.K.),

purchased

International

was from the Boehringer Corp.

Pure c r y s t a l l i n e (250~g)

was

U.K.)

Amersham

was iodinated as p r e v i o u s l y d e s c r i b e d toxin

& METHODS

(LUMAC

spectrometer

392

Rapid Important Paper

a)

25

10 i

o × o

o b

F

E o

0

b)

(9 25 (u (9 (9 lO o

////////~//////////////.'/////////// i 20

fraction I

Fig.

1

Release

of

60

number

I

0

I

| 40 i

50 perfusion

time

i00 (min)

[ 3H]dopamine

from

striatal

J

150

synaptosomes.

S y n a p t o s o m e s w e r e p r e i n c u b a t e d w i t h [ 3 H ] d o p a m i n e and p e r f u s e d w i t h a) n o r m a l medium, or b) medium containing 5 x 10-BM neosurugatoxin for 90 m i n (,////,). P u l s e s (100ul) of D M P P (5 x 10-6M; ~ ) or K + (28 m M , ~ ) r e s u l t e d in r e s p o n s e s i n d i c a t e d by the arrows. after

40 min;

conditions inhibiting

to

Fig.

ib).

those

of

DMPP-evoked

Exposure the

to ~ b u n g a r o t o x i n

neosurugatoxin

['H]dopamine

release.

(5 x l~aM)

experiments, Neosurugatoxin

under

was also

identical

ineffective

in

inhibited

the

Rapid Important Paper binding

of

['H]nicotine

radio-labelled In contrast, to

the same

nicotine

when

but

these

tissue

neosurugatoxin nicotine

to rat brain

abungarotoxin

ligands

(Fig.

were

any effect,

also good

(Fig.

was

tested

2a).

largely

against

2b) s b u n g a r o t o x i n

was w i t h o u t

and DMPP were

membranes

393 L-nicotine

ineffective

at c o n c e n t r a t i o n s of

up

to 10-~M.

[*2sI]Sbungarotoxin

was the most potent

inhibitors

displaced

binding

competitor

up to 10-SM.

[*2sI]abungarotoxin

and

However,

binding.

DISCUSSION

The

presynaptic

terminals

nicotinic

(Chesselet,

nicotinic

receptors

pharmacologically that

antibodies

block

the

synaptosomes was not

(Giorguieff raised

(Futerman

of

our

a

This

et al.,

against

cholinergic

of dopamine

provides

in the brain.

1982;

in our

system

of

Mills

assay

from for

receptor

from

dopamine

release 1984)

We have system

a)

demonstrated

skeletal from but

et

muscle striatal

Sbungarotoxin improved

the

1985)

and

al.,

b) i00

lO0

o ~4 4J co u o~

c.. -,~

50

50

c

c.

I

!

i

i0

9

8

7

6

5

Fig. 2 Effect of drugs and toxins abungarotoxin binding to rat brain

i0

4

log

[inhibitor]

9

8

7

6

5

4

(M)

on a) ['H]nicotine membranes.

and b)

of

characterised

now g r e a t l y

(Rapier

nerve

population

We have p r e v i o u s l y

& Wonnacott,

perfusion

striatal

a

has been p a r t i a l l y

nicotinic

preparations.

synaptosomal

release

functional

1977).

the

facilitation

et al.,

very e f f e c t i v e

sensitivity

facilitation

1984)

[125I]-

Membranes (P2 fraction) were p r e i n c u b a t e d with L-nicotine (O--O), DMPP (O--4), ebungarotoxin (Z~-~) or neosurugatoxin (~ ~) at the final c o n c e n t r a t i o n s indicated for i0 min before addition of radioligand and assay as d e s c r i b e d in the Materials & Methods section. Data from a representative experiment are p r e s e n t e d as a percentage of specific binding; each point is the mean of triplicate determinations, with variance of less than 10%.

394

Rapid Important Paper

clear

repetitive

responses

(Fig.la).

In this

diminished

following

such treatment (Fig. the

ib).

These

effect

under be

previously would

to

employed

(1980)

This

in

cholinergic (1978)

(Mills

been

are

Yamada,

and

DMPP

blood

on

contraction

but

et al.,

1976;

(1975)

pressure

(Hayashi

act at p r e s y n a p t i c

central

nicotinic

varicosities

nicotinic

result

of M i s g e l d on

the

Belleroche

et

al.

intrinsic & Bradford

acetylcholine

x

- evoked

l~TM-abungarotoxin

response

receptors

to

receptor

nicotinic

the

first

function receptors

latter

that

(see

in

of

the

brain,

and &

in the study of the

actions

of

and D M P P - e v o k e d

action

has

been

In this case,

(Hayashi

antagonism

in c h a r a c t e r

release

1982),

er al.,

from

since

receptors

by

supports

reproduced

the toxin may

transmitter

muscarinic

and

(Hayashi

cat

(via

may be ganglionic

sympathetic

Indeed

White,

the

al.,

junction

1982;

by n e o s u r u g a t o x i n report

in

et

in the

facilitate

Cooper,

(Kosuge

inhibited

1984).

acetylcholine

was u n a f f e c t e d is

The

et al.,

&

1979).

transmission

ileum.

(Briggs

receptors

neuromuscular

specifically

ganglionic pig

study

exposure

negative

neosurugatoxin

et al.,

surugatoxin

and

and

the

Ascher

itself

surface)

1.8

nicotinic

at

guinea

present

by

de

nbungarotoxin

at this nicotinic

study

of

blocks without

the brief

effect

although (38%)

slices

of

not

the

muscle

the

without

was

with

than

This clearcut

with

was

surugatoxin

antagonists

Yamada

post-ganglionic

perfusion

is not an antagonist

slices,

striatal

specifically

~Bungarotoxin

blockade

1984).

agreement

neosurugatoxin

myenteric

that the toxin

inhibition

that

ganglia

of

release

1984).

Brown

Hayashi

in

striatal

from

potent

1975;

in

reported

parasympathetic

is

modest

release

It has

whereas

l~TM-~bungarotoxin

a

& Wonnacott,

1982),

x

is greatly

K+-stimulated

effective

that ~ b u n g a r o t o x i n

and b e l o w

DMPP

neosurugatoxin

receptor.

& Wonnacott,

range

to

Continuous

a more

molar agonist

Ca++-dependent,

suggest

conditions.

excitation

reported

['H]dopamine Mills

6

synaptosomes

the

strongly

conclusion

which

the on

in the

to the nicotinic

facilitatory

achieve

seem to c o n f i r m

of

effect

findings

identical

receptor.

with

exposure

nicotinic

expected

to drugs

the response

is without

presynaptic

would

are o b t a i n e d

system

on

1984).

The

neosurugatoxin the

view

that

(see Schmidt

the the

of many

et al.,

1980). In

agreement

inhibits assessment

of

instability is

with

['H]nicotine

clear

the

inhibition

of n e o s u r u g a t o x i n

however,

[~2sI]abungarotoxin

is

parallel

using

Nicotinic

antagonists

to nervous

& Murphy,

nicotine

binding,

al.

brain

in our

membranes the

same are

(Romano

with

we

effect (Fig.

was

on

binding

2);

poor

the

studies.

Quantitative because

of

Marks

of

1982).

of

assays

the What

[3H]nicotine

binding

inhibitors

1980;

neosurugatoxin

et al.,

dilutions

~bungarotoxin

other

that 2a).

difficult

(Kosuge

serial very

find

(Fig.

solution

& Goldstein,

and we find that

in agreement

(1984) membranes

experiments

in aqueous

generally

tissue

1983),

et

to

differential

brain

in

Costa

its

to

performed

binding

Hayashi

binding

and were

neosurugatoxin. of

[3H]nicotine

& Collins,

has little

effect

Neosurugatoxin,

1982; on

~'H]

however,

Rapid Important Paper shows

strong

inhibition

to have

potent

nicotine

and

effect DMPP

are

of

binding

at the

and

nicotine

effective

(Fig. 2b; Marks & Collins,

1982),

data

do

of

distinct

preclude

sites

on

the

binding same

inhibition of n i c o t i n e - e v o k e d abungarotoxin

binding

the

binding

competitors

sites

not

is

395 first site.

for

[3H]nicotine which

to

In contrast,

[*2s I~bungarotoxin

neosurugatoxin

oligomers,

antagonist

shown

although binding

is without effect.

and would

be

Such

[12sI ]~bungarotoxin be

consistent

with

at the

release by an a n t i s e r u m that crossreacts with the

component

from

rat

brain

(Mills

&

Wonnacott,

1984).

I r r e s p e c t i v e of the precise r e l a t i o n s h i p of the b i n d i n g

sites,

consistent with the w i d e l y held view that a b u n g a r o t o x i n

is a poor antagonist of

known

nicotinic

cholinergic

function

in the CNS.

shows c o n s i d e r a b l e p r o m i s e as a potent,

Conversely,

our results

are

neosurugatoxin

selective probe for functional central

nicotinic receptors. ACKNOWLEDGEMENTS We are very grateful to Professor T. Kosuge for the generous gift of neosurugatoxin. This study was supported financially by the Medical Research Council, the Mental Health Foundation and the Tobacco Advisory Council. C.R. is in receipt of a p o s t g r a d u a t e training award from the S.E.R.C. We are grateful to Jenni Miles for p r e p a r a t i o n of the typescript. REFERENCES Ascher P., Large W.A. and Rang H.P. (1979). Studies on the m e c h a n i s m of a c e t y l c h o l i n e antagonists on rat p a r a s y m p a t h e t i c ganglion cells. ~. Physiol. 295, 139-170. Barnard E.A. and Dolly J.O. (1982). Peripheral and central nicotinic a c e t y l c h o l i n e receptors - how similar are they? Trends in Neurosci. ~, 325-327 Briggs C.A. and Cooper J.R. (1982). Cholinergic m o d u l a t i o n of the release of [~H]acetylcholine from s y n a p t o s o m e s of the myenteric plexus. J. Neurochem. 38, 501-508 Brown D.A., G a r t h w a i t e J., Hayashi E. and Yamana S. (1976). Action of surugatoxin on nicotinic receptors in the superior cervical ganglion of the rat. Br. J. Pharmacol. 58, 1 5 7 - 1 5 9 . C h e s s e l e t M.F. (1984). Presynaptic regulation of n e u r o t r a n s m i t - ~ r release in the brain. N e u r o s c i e n c e 12, 347-375 C o n t i - T r o n c o n i B. and R a f t e r y M.A. (1982). The nicotinic cholinergic receptor: c o r r e l a t i o n of molecular structure with functional properties. Ann. Rev. Biochem. 51, 491-530. Costa L.G. and Murphy S.D. (1983). ['H]Nicotine b i n d i n g in rat brain: alteration after chronic a c e t y l c h o l i n e s t e r a s e inhibition. J. Pharmacol. Ex~. Ther. 226, 392-397. de B-'~le~-6"che J.S. and B r a d f o r d H.F. (1978). Biochemical evidence for the presence of presynaptic receptors on dopaminergic nerve terminal. Brain Res. 142, 53-68. Futerman A.H., Harrison R., Lunt G.G. and Wonnacott S. (1982). An immunological approach to the study of presynaptic a c e t y l c h o l i n e receptors.

396

Rapid Important Paper

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