Neurochemical effects of et 495 : Mechanism of action

607

Pharmacological Research Communications, Vol. 13, No. 6, 1981

NEUROCHEMZCAL EFFECTS OF ET 495

: MECHANISM OF ACTION

Hiremagalur, J. Hrishi Keshavan, Nirmal K. Gurbani and Prem C, Dandiya* Department of Pharmacology, S.M.S. Medical College, JAIPUR 302 004, India. * Faculty of Medicine, Ai-Fateh University, Tripoli, Libya. Received in final form 26 March 1981

SU~,~RY Piribedil (ET 495) a predominantly dopamineroic agonist depletes brain 5-HT and lowers 5-HIAA

receptor levels

apomorphine and amphetamine treated rats. It also norepinephrine levels in these animals. It is

lowers

suggested

that the post-synaptic dopamine receptor stimulation piribedil has an inhibitory effect on the

in

by

noradrenergic

and serotonergic neurotransmission in the brain.

INTECDUCT ION Various reports available indicate that piribedil acts on the post-synaptic dopamine (DA) receptors. induces

stereotypy and

In moderate doses, it

behavioural hyperactivity in animals

(Costall and Naylor, 1974; Angrist et al., 1975). It has been found that it has beneficial (Post et al., 1978).

effects

Piribedil

also

in

mental

dilates

artery possibly acting via the pre-synaptic DA (Laubie et al., 1977; Buylaer~, 1977).

0031-6989/81/060607-09/$02,00/0

Thus,

depression

the

femora]

receptors it

shares

©1981 The Italian Pharmacological Society

608

Pharmacological Research Communications, VoL 13, No, 6, 1981

several of the pharmacological

actions of DA and apomorpbine-

another DA agonist.

However, novl t h e r e i s that

growing evidence i n t h e

literature

some of t h e b e h a v i o u r a l and b i o c h e m i c a l e f f e c t s

of

apo-

morphine are mediated via serotonergic system (Ernst, Grabowska e± al., 1973a,b; Scheel Kruger and 1974).

Hasselager,

It is no± clear whether stimulation of

DA

has any modulating or regulatory control on the serotonergic

outflow.

Apomorphine

1967;

pathway putative

is known to elevate

the

5-HIAA levels in the rat and mouse brain indicating an

in-

creased turnover of 5-HT and •ichaluk,

1974).

(Grabowska et al., 1973a; Grabowska

However,

conflicting reports are avai-

lable about piribedil regarding its influence on 5-HT bolism.

Fuxe

Tneta-

(1973) reported a decrease in the 5-HIAA levels

following piribedil.

Therefore

in the present study,

an

attempt has been made to investigate the neurochemical effects of piribedil on 5-HT turnover and its interaction v:ith apomorphine and amphetamine agents which are known to

cause

behavioural syndrome in laboratory animals via DA mechanisms.

~kTPRIALS AND A~:YHODS Albino rats weighing 150-2OO g maintained under normal laboratory conditions with

water ad lib and

conditioned

to

a

12. hour light-dark cycle were employed in the present study. All drug soluticns v:ere made in deicnised v:ater except piribedil which v,as dissolved calculated

in O.O]N HCI and the doses

were

on milligram per kilogram of the free base. Piri-

bedi.l (2.0 mg/kg) was administered 30 minutes

prior %o

the

Pharmacological Research Communications, VoL 13, No. 6, "1981

administration

o£ either apomorphine

mine (5.0 mg/kg). was used.

609

(2.5 mg/kg) or ampheta-

For each combination a set of

six

The animals were observed for 30 minutes

rats

follow-

ing the drug treatment after which they were sacrificed

and

the brain tissues were subjected for biochemical estimations.

Estimation of 5-HT, 5-HIAA and N E l.evel._ss After the behavioural observations, the rats were sacrificed by swift spinal decapitation.

Immediately the skull

opened and the whole brain was removed, weighed and ferred to a frozen plastic box containing acidified butanol and stored at -10°C.

was trans-

lO ml ( purified

The entire procedure

from decapitation to the removal of the brain and its rage in the deep freezer was carried out in less minutes.

)

sto-

than

two

Various brain areas were separated as described below.

A transverse cut is made at the venous sinus to get whole bral hemispheres

along with the olfactory

from the brain stem.

cere-

lobes separating

them

A caudal cut is made at the vermis and the

cerebellum is separated from the medulla and the brain stem the level of the posterior choroid plexus. estimations,

For the

at

biochemical

the method of Haubrich and Denzer (1973) was

emp-

loyed with certain modifications.

Tissue samples :,ere

homo-

genised using a taflon homooeniser

(York Comoany,

in

India)

chilled acidified butanol (lOmM HCI) to give a final tissue concentration of 25-75 mg/ml. minutes at 6000 g.

The samples were centrifuged for

Four ml of the aliquot

was

transferred

50 ml glass stoppered conical centrifuge tube contabning washed n-Heptane and 1.5 ml of O.OIN HCI.

At this stage,

5 to a

i0

ml the

internal standard for 5-HIAA was added (i.0 ug/ml in O.OIN HCI

Pharmacological Research Communications, Vol. 13, No. 6, 1981

610

5-hydroxyindole

acetic acid cyclohexyl ammonium salt A grade,

Calbiochem, California,

USA),

After vigorous

shaking

for

b

minutes to extract the amines, the tubes were centrifuged for 15 minutes at lOOO g. then transferred

A 10 ml aliquot of the organic phase was

to another tube ~or 5-HIAA estimation

friend et a l., 1963).

(

Uden-

A 1.O ml of the acid phase ~,as then tran-

sferred to a 15 ml centrifuge tube. internal standards for norepinephrine

At this stage a mixture and serotonin

(1.O ug/ml

in O.OiN HCI l-norepinephrine HCI, Sigma Chemical Company, Louis, USA;

St

1.O ug/ml in O.OIN HC1 Serotonin creatinine sulphate,

Sigma Chemical Company, St Louis, USA) was added, shaken To this 200 mg of alumina Sayre,

of

(prepared as described by

well.

Anton

and

1964) was added, shaken well and later 1.5 ml of 0.5

Tris-HC1 buffer (pH 8.5)

containing

sodium metabisulphite was added.

O.13 M EDTA

The tubes were then

for 5 minutes to adsorb the catecholamine, minutes a t

1000 g and a 2 . 0 ml p o r t i o n

then transferred

{o a centrifuge

and

13 shaken

centrifuged for 2

of t h e

supernatant

~'as

tube.

Procedure for extraction of norepinephrine The remainder o£ the supernatant aspirated and discarded.

The

liquid

adsorbent

shaking v:ith 2.0 ml of deionised v.ater.

over the alumina ,;:as

was The

then ~:ashed w a s h water

by was

removed by aspiration and the alumina was then shaken for five minutes with l.O ml of O.2N acetic acid to elute the catecholamine.

The 1.O ml p o r t i o n

transferred bed oy Chang

to

a separate

(1964).

of t h e tube

supernatant l i q u i d

for the

assay

of NE as

was

then

descri-

Pharmacological Research Communications, VoL 13, No. 6, 1981

Procedure f o r

@11

extraction of 5-hydroxytryptamine

(5-HT)

The 2.0 ml portion of the supernatant fluid was transferred to a 50 ml conical centrifuge tube containing (salt and water saturated), borate buffer (pH lO.O).

1,5 g of sodium chloride and 0.35M

The mixture was shaken for 5 minutes,

then centrifuged and an aliquot

(5.0 ml) was transferred to

another centrifuge tube containing ml of O.1N HCI. rifugation, of serotonin

6.0 ml of n-butanol

lO.O ml of n-Heptane and

1

After 5 minutes of shaking followed by cent-

1.0 ml of the acid phase was removed for the assay (Bogdanski et al., 1956).

The data were analysed by Student's

't, test.

RESU LTS Piribedil when administered alone did not induce any behavioural effects.

Pre-treatment

with piribedil in apomorphine and amphe-

tamine injected rat.5 induced characteristic and exaggerated

preening

behaviour

reflexes.

and

The bicchemical changes in the levels of 5-HT, 5-H!AA NE in the various parts of the brain are presented

in

Table

cerebe-

Firibedil when given alone depleted 5-HT levels in the llum, medu!]a and whole brain; all

parts of the brain.

lov.ered 5-HT levels;

brain.

rats,

c e r e b e l l u m and whole b r a i n ;

sed i n t h e m e d u l l a a l o n g w i t h and lowered NE l e v e l s

levels except in the cerebe-

in cerebellum,

I n amphetamine t r e a t e d

cerebrum,

in

In apomorphine treated rats, pirJbedil

lowered 5-HIAA

llum and lowered NE l e v e l s

levels

lowered 5-HIAA and NE

I.

piribedil 5-HIA~

a lowering

in cerebellum,

medulla

and

lowered levels

whole 5-H.T

vere i n c r e a -

in the cerebellar

medulla

in

levels

and v.hole b r a i n .

The

P/~3[~,EL3L (2.0) ÷ A P6.b~]R-

5

~,,r. ~. [5.0)

;qR]BED] L (2.0)

o.o3"

(+_.)o.o8

0.253 0.0~

0.446

0.171

0.187 (t)O.O9 £

0.396 o .o6

0.768 (_)o.t 3

0,727 (_+)0.02" 0.0£*

NE

0.444

0.489 0,07

0.219 0.01

1.304 0.02"

0.07 @

4.543 0.03"

2.870 0.02 £

0,216 o.oi £

2.297 0.02 @

0.09 @

1.970

4,020 0.06"

3.985 0.09

1,895 0.01 @

0.06

3.130

NE

* p ~ O.O5 @ P Z 0.01 £ P Z 0.001

1.544 0.09 £

0.09

2.593

2.814 0.07

0,551 0.08

0.962 0.07"

i ,805

3.080 0.02

5-HIAA

0.03 ©

1.424

0.04

0.515

5-HT

Cerebel lure

0.09"

0.:401 0,09

0.524 0.03

Each nroup consisted o[ six rats.

6

AI,:Pi{ETA,'~;E (5,0)

4

m;z:;£ (2.5)

Ar'tA'&RPHINE (2.5)

3

O. 149

0.404 0.09

0,330 (+) 0.06

PIRI3EDIL

2

(2.0}

0.03

0.469

5-H]AA

0.323 (+_)0.02

5-HT

Cereorum and Clfact ory lobes

CC~,:Ti:CLS

bruq Treatment { ~/!,g )

1

P

U

G r o

0.372

1,138 0.06"

1.163 0.01"

0.731 0,01

O.731 0.07

NE

5-HIAA

0.01

0.597

0.657 O.O8

0,707 0.06"

0,561 0.02

0.734 O.O3

NE

2 : 1 , 3 : 1 , 4 : 1 , 5:3, and 6:4 2 : 1 , 3 : 1 , 4 : 1 , 5 : 3 , and 6:4 4:1, 5:3, and 6:4

0.411 0.0~*

0.09 e 0.08

0.212 0.666

0.735 0.798 0,02 @ O,O9

0.431 1.046 0.02 0.03*

0,551 0.633 w 0,004" O. 08

0.393 0.799 0.02 0.O6

5-HT

Whole Brain

NE

2.022 0.436 0.274 0,570 0.08 ~£ 0.02 @ 0.08 £ 0,07

0.'O3@ o.or

0.855

1.004 0.05

2.260 0.02*

1.497 0.G9

1.470 0.08

5-HIAA

between between between

0.204 0.07

0.09'

0.416

0.319 0.08@

1.919 0.07 @

1.580 0.03 @

0.731 0.06

5-HT

~,edulla and Brain stem

=-'.'~ects n'." p ~ r i 3 e d i ! on agomorohine and amphetamine induced changes i n b r a i n 5-HT, 5-HIAA and l e v e l s ( uO/g +_. S.E. Jet Tissue ;,eight )

Table I ~o

(n

PharmacologicalResearch Communications, VoL 13, No. 6, 1981

613

difference being a decrease in 5-HIAA levels in the cerebellum of amphetamine treated rats which was not observed in

apomorphine

treated rats following piribedil.

DISC USS ION Grabowska" (1975) reported that apomorphine has no effect on levels of brain "5-HT, however,

it accelerates the

the

depletion

of

s

5-HT that is" caused by agents which inhibit 5-HT synthesis cating.an increased utilization apomorphine.

o£ brain 5-HT

The effects of apomorphine

indi-

in the presence of

on 5-HT outflow appears

to be due to its primary action on the DA receptor.This

is supp-

orted by the fact that apomorphine

of brain

induced acceleration

5-HT d e p l e t i o n i n H 22/54 p r e - t r e a t e d roperidol,

a D~ a n t a g o n i s t

wska et a l . , ntiated

1973b).

(Grabowska and MichaZuk,

In our s t u d y ,

piribedil

1974; Grabo-

a DA a o o n i s t

pote-

apomorphi.ne induced b r a i n 5-HT d e p l e t i o n and a l s o l o v e r -

ed 5-HIAA l e v e l s brain.

r a t s was a o o l i s h e d by s p i -

in the cerebrum, c e r e b e l l u m ,

These observatJcns

further

i~Scate

medulla and

that

v:hole

the s e r o t o n e r -

gic actions of apomorphine are secondary to DA receptor stJmu]nfinn action. Piribedil does not have any influence on NE levels cerebrum

in

(in apomorphine treated rats) whereas it iov:ered

the the

leveis in other parts of the brain in apomorphine and amphetamine treated rats.

It may be suggested that the NE neurons

be receiving an increase in the impulse infiux induced Oy receptor stimuiation

can DA

leading to an inhibition Jn their activity.

It has been estaOlished that catecholamine

receptor

aoonists

inhibit central DA and NE neurotransmission through

various

614

Pharmacological Research Communications, Vol. 13, No. 6, 1981

mechanisms such as activation of inhibitory autoreceptors~Carlsson, 1975; Kehr et a1.,1972, b).

i977; AghajanSan and B'unney,1977a,

I± ~s hypothesised that piribedil reduces NE

stimulating post-synaptic DA receptors.

release

It is suggested

by also

that piribedil may be reducing serotonergic transmission by its action on post-synaptic DA receptors.

AC KNCYJLEDG ElemENTS Authors are indebted to Les Laboratories Servier, for the general supply of Piribedilo We also wish to acknowledge the help given by Dr S.K.Kulkarni in preparing this manuscript and to Mr Kewal Ram Jeswani for the secretarial assistance.

EEFERE~ ES

Aghajanian, G.K. and Bunney, B.So (1977a). Adv• ,3iochem• Psychopharmacol., 16, 433• Aghajanian, G.K. and Bunney, B.S. (1977b). Arch. Fharmacol. 297, 1. A n g r i s t , B., Thompson, H., Shot,sin, B. and Gershon,S.(1975). Psychopharmacologia 44, 273. B o g d a n s k i , D,F•, P]etscher, A•, B r o d i e , B.B. and U d e n f r i e n d , S. (1956)• J . Pharmacol. Exo. T h e r a p . 117, 82.

Buylae~, "l.A.

(1977). Arch. Pharmaco]...299, 101.

Carlsson, A. (1975). Receptor mediated control of dopamJne metabolites in 'pre-and post-synaptic receptors' eds:Usdin, E. end Nunney, ,.r{. Niarcel Dekker, New Ycrk, 49.

Chang, C. {1964). I n t . J. Neuropharmacol. 3, 643.

Costall, B. and Naylor, R . J .

(1974). Arch. PharmsCO]o28~_5,Tl.

Ernst, A.M. (lo67). Psychophsrm~colcg~a 10, 316.

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615

Fuxe, K, (-1973). Tools in the treatment of parkinson's Disease : Studies in new types of DA receptor stimulating agents in 'Advances in Neurology' eds.Calne, D.B. Raven Press New York, 3, 273, Grabowska, M. (1975). Pharm. Biochem. Behav. 3, 589. Grabowska, M. and f4ichaluk, J. (1974). Pharm. Biochem. Behav. 2, 263. Grabowska, M., Antikiewicz, L., Maj, J. and Michaluk,J.(1973a). Pol. J. Pharmacel. Pharm., 25, 29. Grabowska, M., Michaluk, J. and Antikiewicz, L. (1973b). 3. Pharmaco~. 23, 82.

Eur.

Haubrich, D.R. and Denzer, J.S. (1973). Analyt. ~3iochem.5_~5,306. Kehr, W., Carlsson, A., Lindqvist,M., Magnusson, T. and Atack,C. (1972). J. Pharm. Pharmacol., 24, 744. Kehr, W., Carlsson, A. and Lindqvist,'M. (1977). Arch.Pharmacol. 297, lll. Post, R.~'~., Gerner, R.H., Carman, J.S , Gillin, 3.C., Jimerson, D.G., Goodwin, F.K. and Bunney, W.E. i1978). Arch. Gen.Psychiat 35, 6o9. Scheel Kruger, J. and Hasselager, E. (1974)o Psychopharmacolooia 366, 189. Udenfriend, S., Weissbach, H., and Brodie, B.B. (1963). Methods in Biochemical Analysis, ed. Giick,D. pp 95, Interscience Publishers, New York.