Inhibition by beta-endorphin of dopamine-sensitive anenylate cyclase in rat striatum

Inhibition by beta-endorphin of dopamine-sensitive anenylate cyclase in rat striatum

VoI. 77, No. 1, 1977 INIIIBITION BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS BY BETA-EMDO~PHIN OF DOPAIalNE-SENSITIVE AnENVLATE CYCLASE IN ...

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VoI. 77, No. 1, 1977

INIIIBITION

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BY BETA-EMDO~PHIN OF DOPAIalNE-SENSITIVE AnENVLATE CYCLASE IN RAT STRIATU~

Toshiharu

~1otomatsu,

Martin

and ~ i c h e l Clinical

Research

June

Nabil

Institute

of Nontreal,

H2U IR7

6,1977

SUMMARY. Beta-LPH 61-91 (beta-endorphin) and d o p a m i n e - s t i m u l a t e d adenylate cyclase of rat s t r i a t u m . N a l o x o n e p r e v e n t e d this

Endogenous have

been

taries

isolated

(1-4).

and

inhibiting

vas

deferens

has

some

and

demonstrated cataleptic

peptides and

of or

on

action

endorphins.

An

to

increase

inhibitory

cyclase observed

the

cyclase

MATERIALS

AND

of

intracellar

of

been

4.6.1.1.)

receptors of m o u s e

can

produce

report

(6),

mechanism studies

nucleotides

AMP

pitui-

(beta-endorphin)

and

previous

Recent

cyclic

shown

system

a putative

on

oniate

61-91

a dopaminer~ic

cyclic

of b e t a - L P H

(EC

our

61-91.

of m o r p h i n e

has

effect

adeDylate

The

In

and

contractions

beta-LPH

(5-8).

(endorphins)

brains

to b r a i n

stimulated

Dopamine,

effect

activity

binding

nervous

involvement

activity

animal

central

of b e t a - L P H

the

shown

the

i n h i b i t e d the b a s a l a c t i v i t y in a h o m o g e n a t e effect.

opiate in

ileum,

involvement

suggested of

Dig

catatonia

effect

to

electrically

guinea

the

with

identified

In a d d i t i o n

actions

analgesia

Seidah

Chr#tien

Montreal Received

Lis,

in

in

the

61-91

on

activity

the in

have

cellular

neurotransmitter,

levels

In

tbe

(9,10)

in

rat

the

was

striatum

dopamine-stimulated

(12,13).

we

present

(!i).

adenylate study,

we

dopamine-stimu!ated rat

striatum.

METHODS

M a l e S p r a g u e D a w l e y rats s t r i a t u m w e r e d i s s e c t e d by the

Copyright© 1977by AcademicPress, Inc. Allri~tsolrepro~etioninanyformreserved.

weighing 200-250 g were m e t h o d of G l o w i n s k i and

used. Iversen

442

ISSN 0006-291X

Vol. 77, No. 1, 1977

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

300

E 200

~

100

10-7' 10-6 10-5 I0-4 2x10-4 CONCENTRATION OF •-LPH 61-91 (M) F i g u r e i. E f f e c t of b e t a - L P H 6 1 - 9 1 on the d o p a m i n e - s t i m u l a t e d a d e n y l a t e c y c l a s e a c t i v i t y in a h o m o g e n a t e of rat s t r i a t u m . The r e a c t i o n m i x t u r e s c o n t a i n e d 200 uM d o p a m i n e . In the a b s e n c e of d o p a m i n e and b e t a - L P H 61-91, 370 ± 2 p m o l of c y c l i c AMP was f o r m e d . The i n c r e a s e in c y c l i c AMP a b o v e this b a s a l l e v e l is p l o t t e d . The d a t a r e p r e s e n t the m e a n ~ SE f r o m triplicate determinations. * P < 0.05, ** P < 0 . 0 1

(14). The s t r i a t u m w e r e h o m o g e n i z e d in 13 v o l u m e s of 2mM t r i s m a l e a t e b u f f e r (pH 7 . 4 ) - 2 m M EGTA. A d e n y l a t e c y c l a s e a c t i v i t y was d e t e r m i n e d by a m o d i f i c a t i o n of the m e t h o d of S o l o m o n et al. (15). E a c h tube c o n t a i n e d 8 0 m M t r i s - m a l e a t e (pH 7.4), 5 m M M g C I 2 , 2 0 m M c r e a t i n e p h o s p h a t e , I0 u c r e a t i n e p h o s p h o k i n a s e , imM ~ I p h a - 3 2 p ] - A T P (1-2 x i0 ° cpm), i m M cAMP, 1 0 m M t h e o p h y l l i n e and h o m o g e n a t e c o n t a i n i n g 200 ug of p r o t e i n in a f i n a l v o l u m e of I00 ul. I n c u b a t i o n was for 5 m i n at 30°C. C y c l i c AMP f o r m e d was p u r i f i e d u s i n g a c o m b i n a t i o n of D o w e x 50 and a l u m i n u m o x i d e c h r o m a t o g r a p h y . D a t a are p r e s e n t e d as p m o l e s of c y c l i c AMP f o r m e d per mg p r o t e i n per 5 min. B e t a - L P H 6 1 - 9 1 was i s o l a t e d and p u r i f i e d as d e s c r i b e d p r e v i o u s l y (4). For s t a t i s t i c a l a n a l y s i s of the r e s u l t s , N e w m a n K e u l s m u l t i p l e r a n g e test (16) was used. RESULTS Beta-LPH eyclase 10 -5,

activity

10 -4 and

increased was

61-91

in a h o m o g e n a t e

2 x 10-4M

inhibition

achieved

inhibited

of

of rat

beta-LPH cAMP

at a b e t a - L P H

dopamine-stimulated

61-91

production.

61-91

striatum caused Fifty

concentration

10-5M.

443

(Fig.

adenylate i).

10 -6 ,

progressively % inhibition of

just

over

Vol. 77, No. 1, 1977

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 500

4400 E 300

I

:E

200 I1.

u

I

100

!

DOPAMINE

I-LPH 61-91

BASAL

DOPAMINE fJ-LPH 61-91

F i g u r e 2. E f f e c t of b e t a - L P H 6 1 - 9 1 on the b a s a l and d o p a m i n e s t i m u l a t e d a d e n y l a t e c y e l a s e a c t i v i t y in a h o m o g e n a t e of rat striatum. 200 u M b e t a - L P H 6 1 - 9 1 and i00 uM d o p a m i n e w e r e used. D a t a r e p r e s e n t the m e a n s ± SE f r o m t r i p l i c a t e d e t e r m i n a t i o n s . * P < 0.01: b a s a l vs b e t a - L P H 6 1 - 9 1 ** P < 0.01: d o p a m i n e vs d o p a m i n e + b e t a - L P H 61-91.

In a d d i t i o n enzyme,

beta-LPH

of b a s a l 61-91

61-91

adenylate

reduced

activity

inhibitory

cyelase

(Fig.

2 x 10-4M

naloxone

of a d e n y l a t e partially

cyclase

prevented

by

10 -6

dopamine-stimulated

but

(Fig.

to a b o u t

effect

adenylate

on the

a slight

activity

stimulated 3) o

effect

showed

cyclase

basal

The

to the

90%

significant 2). of

of b e t a - L P H

activity

was

completely and

10-5M

the i n h i b i t i o n

by

inhibition

2 x 10-4M beta-LPH the

control

61-91

value.

on the d o p a m i n e -

prevented prevented

by n a l o x o n e the

of b e t a - L P H

inhibition

61-91

IO-4M beta-LPH

and

61-91

(P

action

of

" 0.01)

DISCUSSION Our beta-LPH opiate

61-91

results

suggest

on d o p a m i n e r g i c

receptors

in c y c l i c

~P

a direct

antagonistic

neurones.

The

regulation

is also

results.

444

idea

of i n v o l v e m e n t supported

by our

of

Vol. 77, No. 1, 1977

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

z

t

300

E

Q.

o~ 200 =E

~ I00 Is,

I

/V~

0

I

I

I

I

10-e

I0 -7

I0-6

fO-5

I,

I0-'~

CONCENTRATION OF B-LPH 61-91 (M)

F i g u r e 3. E f f e c t of b e t a - L P H 61-91, a l o n e or in c o m b i n a t i o n w i t h i00 uM n a l o x o n e , on the d o p a m i n e - s t i m u l a t e d adenylate c y c l a s e a c t i v i t y in a h o m o g e n a t e of rat s t r i a t u m . Reaction m i x t u r e s c o n t a i n e d i00 uM d o p a m i n e . In the a b s e n c e of d o p a m i n e , b e t a - L P H 6 1 - 9 1 and n a l o x o n e , 379 ~ 2 p m o l of c y c l i c AMP was formed. The i n c r e a s e in c y c l i c A M P a b o v e this b a s a l l e v e l is p l o t t e d . D a t a r e p r e s e n t the m e a n s ~ SE f r o m t r i p l i c a t e determinations.

In a p r e v i o u s effects

of L - D O P A

Apomorphine, been

shown

rats

(17).

to a b o l i s h

observations. as

5 nmol

present The

per

The

it w e r e

evenly

present

61-91

over

adenylate

fluid

results

given

of

are

the

activity

dopamine

effect

this

that

for

in v i t r o .

445

the

the

The

61-91

was

also in

determined

concentration

be a b o u t

cerebrospinal

has

earlier

catalepsy

concentrations

changed

receotor,

with

dose would

in 1 ml of

in rat.

of b e t a - L P H

consistent 61-91

the a n t a g o n i s t i c

catalepsy

intraventricularly.

following

shows

demonstrated

61-91-induced

of b e t a - L P H

1 uM s i g n i f i c a n t l y cyclase

we

cataleptic

distributed

experiment

(6),

blocker

the

ED50

rat w h e n

in c e r e b r o s p i n a l if

on b e t a - L P H

a specific

The

report

5 uM,

fluid.

of b e t a - L P H

dopamine-sensitive

Vol. 77, No. 1, 1977

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Morphine-like the s t i m u l a t i o n by rat brain not observe

any

change

(24). of basal

of cyclic

could be p r o b a b l y

explained

The i n h i b i t i o n in rat s t r i a t u m

is less

showed

cells.

This

opiate

receptors

Lob et al.

beta-LPH

61-91

symptoms

such

as a k i n e s i a

dopaminergic

neurones

may play

and related

has been

of a n t i p s y c h o t i c cyclase

have been

of a d o p a m i n e r g i c suggested

endorphins

and

(24).

activity

and N i e r e n b e r g

to the lower

the i n h i b i t o r y

rigidity

Parkinson's involves

a pathophysiological

x glioma

density

effect

of

Extrapyramidal

are k n o w n syndrome

to

with

the h y p o f u n c t i o n pathway

role

of

cells.

in rat striatum.

and m u s c u l a r

(19).

akinesia of

Endorphins

in P a r k i n s o n ' s

disorders. of a d o p a m i n e r g i c

extensively drugs

discussed

in rat

mechanism

striatum

(20,21).

(22).

in the b r a i n has

in the p a t h o g e n e s i s

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

in the b r a i n

mechanism

446

effects

adenylate

Hyperactivity therefore

been

of s c h i z o p h r e n i a .

of d e c r e a s e d

of some s c h i z o p h r e n i c

in schizo-

Inhibitory

on the d o p a m i n e - s e n s i t i v e

shown

indicate

of the basal

in the n i g r o - s t r i a t a l

to he i n v o l v e d

Our results

used

homogenate

than in the hybrid

symptoms

The i n v o l v e m e n t phrenia

in methods

by Klee

in

difference

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

may be due

release

decrease

This

brain

did

(by m e t h i o n i n e - e n k e p h a ! i n )

activity

mechanism.

as the m a i n

61-91

also reported

on d o p a m i n e

61-91.

formation

AMP by morphine.

significant

of w h o l e

inhibition

difference

and r i g i d i t y

hut

AMP

however,

of cyclic

that o b t a i n e d

cyclase

a dopaminergic

syndrome

instead

than

(18)

(24),

by the d i f f e r e n c e s

in the s t r i a t u m

Recently

therefore,

a slight

to inhibit

of cyclic

and Roy

production

by b e t a - L P H

a marked

adenylate

hybrid

involve

reported

AMP by b e t a - L P H

by the use of rat s t r i a t u m

of the basal

Collier

w o r k we report

production

(i0), who

already

by E 1 and E 2 p r o s t a g l a n d i n s

homogenate

In our present basal

drugs were

activity

patients.

of

Gunne

Vol. 77, No. 1, 1977

et al.

(23),

the auditory contradict

however,

reported

hallucination

our

to elucidate

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

speculation.

the real

a benefical

effect

in schizophrenia. More

extensive

pathophysiological

of naloxone

This studies

role

result will

on

seems

to

be required

of endorphins.

ACKNOWLEDG!fENTS. We thank Dr P. Hamet and Dr D. Franks for advice concerning adenylate cyc!ase assay and Dr K. Izumi and Dr A. Barbeau for a d e m o n s t r a t i o n of rat brain autopsy. The s e c r e t a r i a l help of ~rs D. ?larcil is appreciated. REFERENCES i. 2. 3 4 5 6 7 8 9. i0. ii. 12. 13. 14. 15. 16. 17.

18. 19. 20. 21. 22.

23. 24.

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