Apomorphine is a potent inhibitor of type 2A protein phosphatase in rat brain

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 176, No. 2,1991

Pages 737-740

April 30,1991

APOMORPHINE IS A POTENT INHIBITOR OF TYPE 2A PROTEIN PHOSPHATASE IN RAT BRAIN

SHINJIRO KAWAI Laboratory of Biology, Osaka Dental University Hirakata , Osaka 573, Japan Received March 18, 1991

SUMMARY: Effects of five kinds of dopamine agonists on the activity of type 2A protein phosphatase in rat brain were studied. Apomorphine and SKF-38393 reduced the enzyme activity considerably and their effects were further enhanced in the presence of 10 ~M Mn2+.Also,6,7-ADTN slightly inhibited the activity.The present results suggest that type 2A protein phosphatase in the brain is possibly involved in dopamine mediated protein phosphorylation functions. © 1 9 9 1 A c a d e m i c Press, Inc.

In their

nervous systems,the regulation of related

protein

factors has been recognized to play an

phosphatases

and

important role

in

nervous functions(l-3). However, in contrast to advances in the

role

of

protein

kinases

in

multiple

understanding

signal

transduction

systems(4,5),the involvementof protein phosphatase systems remainsto be thoroughly investigated. We have proposed that such experiments require a

suitable modulator

for protein phosphatase that is directly associated with i t s physiological functions.

In order

effects of reagents

to device a modulator of this type,

we examined the

used in neurobiological studies on protein phosphatase

activity in rat brain. The agonist

present

s t u d y shows that

apomorphine, a

used to treat Parkinsonism(6) which is known to

typical

sensitive adenylate cyclase(7),is a potent inhibitor of type 2A phosphatase

in

dopamine

promote dopamine protein

rat brain. Also, we found that another dopamine agonist,

SKF-38393, considerably

inhibits the enzyme activity and

that

6,7-ADTN

slightly reduced i t . These results show that a number of dopamine agonists can

inhibit

the activity of type 2A protein phosphatase

suggesting that

these

in

the

brain,

reagents can modulate dopamine mediated protein

phosphorylation in the nervous systems. Materials and Methods Chemicals: ( ± )2-Amino-6,7-dihydroxy-l,2,3,4-tetrahydronaphthaleneHBr (6,7-ADTN),R(-)apocodein HC1, R(-)apomorphine HCI(APO), R(-)2,11-dihydroxy-

737

0006-2913
Vol. 176, No. 2, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10-methoxyaporphine HCl(morphothebaine), and 2,3,4,5-Tetrahydro-7,8-dihydroxyl-iH-3-benzazepine HCI (SKF-38393) were purchased from Reseach Biochemical,Inc.(MA,USA). T h e s e compounds w e r e dissolved in 20% dimethylsulfoxide(DMSO) to the concentration of i00 mMwhen they were used. Okadaic acid and calyculin A were purchased from Wako Chemical(Osaka,Japan). Preparation of t y p e 2A protein phosphatase: Type 2A Protein phosphatase was obtained from the 100000xg supernatant(cytosol) of Wistar rat brain homogenate, which had been prepared basically according to refs.8 and 9. Fractions containing the enzyme were subsequently purified through successive column chromatographies over DEAE-cellulose(DE-52), DEAES e p h a r o s e CL6B, c a s e i n - c o n j u g a t e d A f f i G e l 10 a n d f i n a l l y , B i o G e l A 1.Sm . The e n z y m e a c t i v i t y was e l u t e d i n a s i n g l e p e a k f r o m B i o G e l A 1.5m, a t a position corresponding to about 150-kDa molecular weight, and then stored at -60"C until used. Assay of the enzyme activity: To t h e r e a c t i o n m i x t u r e : 15 Z 1 o f enzyme s o l u t i o n a n d 20 ~ 1 o f 250 mM H e p e s - b u f f e r e d solution(pH 7.5)containing the indicated concentrations of reagents, 15 ~ 1 o f p h o s v i t i n ( 1 5 m g / m l ) ( S i g m a ) , which had been once dissolved i n 100 mM EDTA a n d t h e n d i a l y z e d a g a i n s t an extensive volume of distilled w a t e r , was a d d e d t o s t a r t t h e assay. After incubation f o r 20 min a t 3 5 " C , 10 ~ 1 o f i c e - c o l d 60% p e r c h l o r i c acid was added to the aliquot and kept for one minute on ice. The a l i q u o t was centrifuged a t 14000 rpm f o r o n e m i n u t e t o o b t a i n t h e s u p e r n a t a n t , w h i c h was t h e n a d d e d w i t h 440 Z 1 o f M a l a c h i t e g r e e n s o l u t i o n ( 1 0 ) containing 20 mM o f EDTA a n d centrifuged a g a i n a t 14000 rpm f o r o n e m i n u t e . The r e s u l t i n g supernatant was i n c u b a t e d f o r 20 min a t 30"C, a n d t h e n i t s absorbance at 650 nm was m e a s u r e d to determine the amounts of phosphate. To e v a l u a t e the colorimetrical influence of compounds and ions used in this assay, the absorbance of the reaction mixtures containing 1 . 0 nmol o f p h o s p h o r i c acid in place of enzyme s o l u t i o n was m e a s u r e d i n t h e presence of various concentrations of reagents.None of the following: the tested dopamine agonists at the concentration r a n g e o f 0 . 0 2 - 2 . 0 mM,4%DMSO, 10 ~ M o f o k a d a i c acid and calyculin A, a n d 1 mM o f t e s t e d m e t a l i o n s h a d a n y e f f e c t on t h e phosphate determination. Results The about 0.5

activity

the

nN o k a d a i c and

mercaptoethanol.

acid

(no reagent)

1

control

was p r e s e n t further

10% a t

no e f f e c t or

These have a

relative

not require

eluted

at

inhibited metal

ion

in the presence

was c o n f i r m e d

activity

that

This

concentrations

by for

of

2-

t h e enzyme i s

to

the

metal

was a d d i t i o n a l l y

each concentration

ions,the

activity

in the presence suggested effect

hand,

another

although

the addition

o f APO h a d no e f e c t that

enhanced

Mn2+, t h e a c t i v i t y

of APO(Fig. 1),

On t h e o t h e r

about when

50% Mn2+

decreased

Mn2+ i t s e l f of

of

10 ~ M

a had Mg2+

on the enzyme activity. dopamine

o n t h e enzyme a c t i v i t y , s o

738

control

of apomorphine(APO).

~ M o f APO was e n o u g h t o s u p p r e s s effect

w i t h APO. W i t h 10 Z M

inhibitory

o f t h e enzyme

o f APO w i t h o u t

markedly;1

activity.

results

similar

A,did

it

study,which

was c o m p l e t e l y

freeze-thawing

of various

concentrations

on the activity.

10 ~ M Ca 2+

calyculin after

this

2A1(2,8,11,12).

shows the

together

for

filtration,

properties,

in the presence

increasing

the

o r 0 . 2 nM

From t h e s e

t h e enzyme d e c r e a s e d of

in gel

was s t i m u l a t e d

phosphatase

Figure

With

the enzyme prepared

150-kDa fraction

activity,

protein

of

and Discussion

agonists

may

we e x a m i n e d t h e

V o l . 1 7 6 , N o . 2, 1991

effects

of

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

several

other

reagents:

tetrahydronaphthalene(6,7-ADTN),

2-amino-6,7-dihydroxy-l,2,3,4-

apocodein,

2,11-dihydroxy-10-methoxy-

aporphine(morphothebaine) and 2,3,4,5-tetrahydro-7,8-dihydroxy-l-phenyl-IH3-benzazepine(SKF-38393).

As shown in the f i g u r e s , l i k e APO, 6,7-ADTN and

SKF-38393 inhibited the enzyme a c t i v i t y . However, 6,7-ADTN was

far

e f f e c t i v e than APO,with 2.0 mM 6,7-ADTN decreasing the a c t i v i t y

about

(Fig.2).

On the

considerably; the

other

50 g M

h a n d , SKF-38393 reduced the

enzyme a c t i v i t y

inhibitors were stimulated by I0 gH

50%

inhibition,a

higher concentration than that of APO was required. The

these

40%

SKF-38393 reduced the enzyme a c t i v i t y to about

control l e v e l ( F i g . 3 ) . However,for the same level of

fold

less

50-

effects

Mn2+,and Hg2+ and

Ca2+

of

also

had no e f f e c t , similar to the case of APO. In contrast, apocodein and morphothebaine had no e f f e c t on

the enzyme

a c t i v i t y at the concentration range of 0.02-2.0 mMand s t i l l f a i l e d to show an e f f e c t

e v e n in the presence of 10 gH

The

present

SKF-38393

or

phosphatase form

of

would

study

showed that

6,7-ADTN inhibits

Nn2+.

administration

the

activity

of

of rat

apomorphine, brain

protein

2 A I . Since protein phosphatase 2AI is believed to be a

the type 2A protein phosphatase family (2,8),the present apply

to

other

Apomorphine(7) and sensitive

adenylate

members

of

this

family

SKF-38393(13) have been shown to

of

cyclase at low concentrations. These facts

phosphorylation

such as that

of

results enzymes.

stimulate

present r e s u l t s suggest that apomorphine and SKF-38393 f a c i l i t a t e mediated protein

major

dopamine and

the

dopamine

DARP-32(14)in nervous

systems.The present study thus indicates that a number of dopamine agonists may possibly contribute to signal transduction systems,acting to

100, ~

8o

-°

80~, ~

6O

'~

60

<

/ 4oL

>~ 40

-~

IOC~9

'=

e~

.

.

.

100q 80

---o

...........

~

=

=>

60

o~

40

o ~ o

20

20 rr

nr

E

i

iQ

.

0

modulate

10

20

Apomorphine ( p M )

,

0 ~

i

1 6,7-ADTN (mM)

03 5KF-38393 ( m M )

Fig. 1. I n h i b i t i o n of p r o t e i n phosphatase a c t i v i t y by Apomorphine. ( O ) r e a g e n t o n l y , ( O ) w i t h 10 zM Mn2+. Fig.2.

I n h i b i t i o n of p r o t e i n phosphatase a c t i v i t y by 6,7-ADTN. ( O ) r e a g e n t o n l y , ( O ) w i t h 10 /~M Mn2+.

Fig.3.

I n h i b i t i o n of p r o t e i n phosphatase a c t i v i t y by SKF-38393. ( O ) r e a g e n t o n l y , ( O ) w i t h 10 zM Mn2+. 739

1~

Vol. 176, No. 2, 1991

protein

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

phosphorylation

pharmacological modulating

aspects

effects,their

phosphatases regulation investigation

and of

the protein

functions.This of

these

will

agents.

possible influence effects

cast a To

phosphatases

in

further

on o t h e r

of dopamine and rat

new

its brain

light

on

confirm

types

of

derivatives are

now

the these

protein on

the under

in our l a b o r a t o r y . References

1. K l e e , C . B . , D r a e t t a , G . F . , a n d H u b b a r d , M . J . ( 1 9 8 8 ) i n Advances i n Enzymology and R e l a t e d Areas of M o l e c u l a r B i o l o g y ( M e i s t e r , A . , E d . ) p p 1 4 9 - 2 0 0 , An I n t e r s c i e n c e P u b l i c a t i o n , John W i l l e y & Sons,New York 2. Cohen,P. (1989) A n n u . R e v . B i o c h e m . 5 8 , 4 5 3 - 5 0 8 3. C o h e n , P . , and Cohen,P.T.W.(1989) J . B i o l . C h e m . 264,21435-21438 4. N i s h i z u k a , Y . ( 1 9 8 8 ) Nature 334,661-665 5. S h e n o l i k a r , S . ( 1 9 8 8 ) FASEB J . 2 , 2 7 5 3 - 2 7 6 4 6. L a l , S . ( 1 9 8 1 ) i n Apomorphine and O t h e r Dopaminomimetics ( C o r s i n i , G . , a n d Gesa,G.L.,Eds.) vol.2,ppt-ll,Raven P r e s s , New York 7. K e b a b i a n , J . W . , P e t z o l d , G . L . , a n d G r e e n g a r d , P . ( 1 9 7 2 ) Proc. N a t l . A c a d . S c i . U.S.A.69,2145-2149 8. T u n g , H . Y . L . , A l e m a n y , S . , a n d C o h e n , P . ( 1 9 8 5 ) E u r . J . B i o c h e m . 1 4 8 , 2 5 3 - 2 6 3 9. Okada,M.,Owada,K., and Nakagawa,H.(t986) Biochem.J. 239,155-162 1 0 . K o d a m a , T . , F u k u i , K . , and K o m e t a n i , K . ( 1 9 8 6 ) J , B i o c h e m . 9 9 , 1 4 6 5 - 1 4 7 2 l l . B i a l o j a n , C . , and T a k a i , A . (1988)Biochem.J. 256,283-290 12.Ishihara,H.,Martin,L.,Brautigan,D.L.,Karaki,H.,Ozaki,H.,Kato,Y., Fusetani,N.,Watabe,S.,Hashimoto,K.,Uemura,D.,and Hartshorne,D.J.(1989) Biochem. Biophys. Res. Commun.159,871-877 1 3 . A n d e r s e n , P . H . , and J a n s e n , J . S . ( 1 9 9 0 ) E u r . J . P h a r m a c o t . 1 8 8 , 3 3 5 - 3 4 7 1 4 . W a l a a s , S . I . , A s w a r d , D . W . , and G r e e n g a r d , P . ( 1 9 8 3 ) N a t u r e 301,69-71

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