Pharmacological Research Communications, Vol. 9, No. 8, 1977 755

EFFECT OF CHOLINE, PHOSPHORYLCHOLINE AND DIMETHYLAMINOET]L4NOL

ON BRAIN ACETYLCHOLINE LEVEL IN THE RAT.

1 Felici-ta Pedata, Andrzej Wieraszko and Giancarlo Pepeu

Department of PharmacoloKy, University of Florence,

Florence, Italy. Received 13 June 1977 SUMMARY

The effect of the administration of choline (Ch), phospho-

rylcholine (PCh) and dimethylaminoethanol (DMAE) on acetylcho-

line (ACh) level in the cerebral Cortex and the caudate nucleus

was investigated in rats killed by focussed microwave radiations.

Ch administered i.p. or i.v. up to the dose of 120 mg/]{g caused

neither behavioural effects nor changes in ACh level. Similarly

no effects were detected following administration of ecuimolar

doses of PCh or DI:LkE. PCh (228 mg/K~_ i.p.) exerted a partial

antagonism toward the decrease in striatal ACh induced by intra-

ventricular administration of hemicholinium (HC-3). The intra-

administration of PCh, Ch but not of DI, LkE also anta-

effect of HC-3 on striatal ACh level but not on

ventric ular

gonized the

cortical ACh.

INTRODUCTION

Cohen and Wurtman (1976) demonstrated that ACh concentrations

in the whole rat brain or in various brain regions vary with die-

tary Ch consumption. Haubrich et ai.(1976) showed that intraven-

- - - j I _ _ I i m - - | i I I , - - . _ - - . . . . . . . . .

1present address: Nencki Institute of Experimental Biology,

Polish Academy of Sciences, Dept. of Biochemistry of Nervous

System a n d Muscle, Warszawa 22, Poland.

756 Pharmacological Research Communications, Vol.. 9, No. 8, 1977

tricular, intravenous, intracarotid or intraperitoneal administra-

tion of large doses of eitller Ch or DMAE brings about a marked

increase in the ACh content of the caudate nucleus of rats fed on

a routine laboratory diet. The possibility of increasing brain

ACh content by administration of ACh precursors is of considerable

experimental interest and could have a clinical significance

(Davis et al.,1975). However the results reported by Haubrich et

a1.(1976) are at variance with previous observations (Pepeu et al.,

1960) which showed no increase in rat brain ACh following either

a single i.p. or repeated oral administrations of DMAE or Ch.

This discrepancy prompted us to reinvestigate the effect of the

putative ACh precursors on brain ACh level in the rat. In this

investigation we also studied PCh another precursor for ACh syn-

thesis according to Berry and Stotz (1956) and Cheney et a1.(1975).

MATERIALS AND METHODS

Male Wistar rats (150 - 200 ~) were used for experiments

where drugs were administered by i.p. or i.v. injection. Smaller

rats (I00 - 120 g) were used for the intraventricular injections

which were carried out under light ether anaesthesia according to

the procedure described by Domino et ai.(1973). A microlitre si-

ringe and a nedle with a stop were used for these injections

(depth 4 -4.5 mm, 1.5 mm posterior and 1.5 mm lateral from the

exposed bregma). The rats usually recovered from the anaesthesia

after 3 - 4 rain.

After drug administration the rats were killed at the time

stated in the results by focussed microwave irradiation according

to the procedure described by Guidotti et ai.(1974). A commercial

microwave unit adapted by Medical Engineering Consultant (Lexing-

ton Mass. ,U.S.A.) was used. The microwave output was 1.3 kW at

2.45 GHz and the exposure time was 3 sec. The frontal cortex and

the caudate nucleus were dissected from the brain. ACh was ex- !

tracted in a citrate buffer, pH 3.5 (Beanl and Bianchi,1968) and

Pharmacological Research Communications, Vol 9, No 8, 1977 757

the content of the extracts was determined by bioassay on the frog

rectus abdomini s.

DMAE was supplied by Riker Laboratories Inc. as the p-acetyl-

aminobenzoate under the trade mark name of Deaner; PCh vsas sup-

plied by Geigy as chloride. The other drugs used were from corn- / /"

mercial sources. All~drugs were dissolved, in saline or distilled • t

water and ' i n j e c t e d i n a volume neve r exceedin~,r 0 .5 ml.

RESULTS

When Ch was injected i.v. or i.p. at doses of 30, 60 and 120

mg/Kg and the rats were killed~ 20~40 or 60 min later, no behav-

ioural changes were observed ~or Were ACh levels in the caudate

nucleus and in the Cerebral cortex increased. Similarly no effects

were detected following administration of equimolar doses of PCh

and D~LAE.

Table 1

Effect of choline (Ch), phosphorylcholine (PCh) and dimethyl-

aminoethanol (DI, LAE) on brain ACh level.

m ~ n n n n n I m I IIII I i I

Treatment Dose N o ACh level

mg/Kg i.p. of rats nmol/g + S.E.

cortex caudate n. | i ml m

Saline - 34 20.6 + 1.0 66.2 + 3.2

Ch 120 4 18.7 + 0.9 65.0 + 4.0

PCh 226 4 1 6 . 2 + 0 . 9 6 6 . 9 + 2 . 3

DMAE 2 3 3 4 2 1 . 7 + 0 . 7 6 7 . 8 4, 2 . 0

Scopolamine 0.5 6 11.3 + 1.6 + 46.3 + 4.3 +

Ch + 120 4 11.5 + 2.4 + 50.I + 1.4 +

Scopolamine 0.5 -- --

PCh + 226 2 9 . 8 5 0 . 4

S~opo famine 0.5

- - _ n mUll I n ¿ u m I - - _ . . . .

The rats were killed 40 rain after the administration of Ch and its

precursors and 30 rain after scopolamine. ÷

This value differs from saline with P<O.OI

758 Pharmacolog/cal Research Communications, Vol. 9, No 8, 1977

Table 1 s h o w s the ACh levels 4 0 rain after the i.p. injection

of the higluest dose tested of Ch, PCh and D~,IAE. At that time Hau-

brich et a1.(1976)observed the peak effect of Ch and DMAE. It

also shows that neither Ch or PCh significantly antagonized the

decrease in ACh level induced by scopolamine a ~¢ell kno~vn depletor

of brain ACh (Giarman and Pepeu, 1964).

We also attempted to antagonize the effect of intraventricular

administration of hemicholinium (HC-3) by intraperitoneal injection

of Ch, PCh or DI:DiE (Table 2). The intraventricular administration

of HC-3 was followed I0 rain later by light tremors, muscular twit-

ches and ataxia. No death occurred.

Table 2

Effect of pretreatment with Ch, PCh and DMAEiop.

on brain ACh level of rats injected intraventri-

cularly with hemicholinium (HC-3 25 ug per rat).

Treatment Dose N o ACh level

rag/KS i.p. of rats nmol/g + S.E.

cortex c audate n.

Saline - 26 22.7 + i.i 68.2 + 1.9 o o+

HC-3 - i0 13.6 + 1.4 34.5 + 2.4

Ch + 120 o o 3 1 1 . 5 + 2 . 0 3 7 . 6 + 4 . 4

H C - 3 - - - - -

PCh + 2 2 6 o o+ 7 1 3 . 8 + 1 . 2 4 1 . 9 + 2 . 1

HC - 3 - - - - -

DMAE + 2 3 3 2 7 . 6 3 5 . 4

HC-3

These values differ from saline with P < 0.01

+These values differ with P < 0.05

HC-3 was injected I0 mln after the pretreatment. The rats were

killed 30 rain later.

Pharmacological Research Communications, Vol. 9, No. 8, 1977 759

Thirty rain after HC-3 the ACh content showed a ~D% decrease

in the cerebr, al cortex and a 48% in the oaudate nucleus. 0nly

PCh at the dose of 226 m~/](g exerted a martial but statistically

significant antagonism to~,/ard the effect of IIC-3" on striatal ACh

level since the decrease in striatal ACh was limited to 38%.

However Ch or PCh administered intraventricularly together with

HC-3, martially anta.gonized the effect of HC-3 on striatal ACh

levels but not on cortical ACh. PCh appeared to be slightly more

active than Ch. On the other hand DMAE was completely ineffective.

These results are shovrn in table 3.

Table 3

Effect of the simultaneous intraventricular administration of

hemicholinium (HC-3 25 ug), Ch, PCh and DHAE on brain ACh level.

Treatment Do se N o ACh level

mg/rat of rats nmol/~ + S.E.

cortex caudate n.

Saline - 26 22.7 + I.I 68.2 + 1.9 O

H C - 3 - 1 0 1 3 . 6 + 1 . 4 3 4 . 5 + 2 . 4

C h O . 3 + 7 1 4 . 3 + 1 . 5 4 3 . 3 + 1 . 7 +

H C - 3 - - " - -

P C h , + 0 . 6 6 1 5 . 6 + 2 . 3 5 0 . 8 + 2 . 3 +

H C - 3 - - "

D r,~LE + 0 . 6 2 1 0 . 0 2 8 . 2

H C - 3

. . . . . ~ " . . . . . . n i lm n

The rats were killed 30 min after HC-3 @

This value differs from saline with P K 0.01

+These values differ from HC-3 with P < 0.05

DISCUSSION - - ,|

The concentrations of brain ACh found by bloassay in the

present paper are similar to those obtained by gas-chromatogra-

760 Pharmacological Research Communications, Vol. 9, No. 8, 1977

phic methods coupled with microwave irradiation of the rats

(Schmidt et al.,1972; Racagni et al.,1975; Modak et al.,1975).

The method used in this paper allowed us to detect a decrease in

ACh content induced by the administration of scopolamine or HC-3.

This decrease was similar to that previously reported by Giarman

and Pepeu (1964), Domino and Olds (1972) and Domino et ai.(1973).

Therefore there is no apparent reason to suppose that changes in

ACh level following the intraper?'itoneal administration of Ch and

its precursors would have gone undetected. Moreover an antagonism

between HC-3 and Ch administered intraventricularly was also seen,

confirming previous results of Slater (1968).

It is however difficult to explain the reasons for the dis-

crepancy between the results obtained by Haubrich et ai.(1976) by

means of an electrophoretic and radiochemical method and our

results. Differences in rat strains and in the diet should per-

haps be taken into account. A further complication associated with

radiochemical techniques might be due to incomplete electrophoretic

separation between ACh and Ch particularly in the presence of

large amounts of Ch. This problem has been recognized before and

possible solutions presented (Ladinsky et ai.,1974).

Barry and Stotz (1956) claim that PCh could provide phosphate

for the ATP necessary for ACh synthesis. Whether this or a better

penetration into the brain, in comparison to Oh, is the reason for

its efficacy is still matter of debate.

The observation that the antagonism of PCh and Ch toward

HC-3 can be seen in the caudate nucleus but not in the cerebral

cortex is further evidence that ACh turnover rate is higher in

the caudate nucleus than in the cortex (Trabucchi et ai.,1975).

In conclusion we have sho~au that the acute administration

of exogenous Ch and its precursors to increase brain ACh con-

centration is of limited value. On the other hand the high

affinity Ch uptake by the cerebral tissue (Yamamura and Snyder,

Pharmacological Research Communications, VeL 9, No. 8, f977 761

1973) is easily saturated and therefore should represent an

obstacle to large increase in ACh level•

AKN0 WLE D GE ME NT S

The present work was supported partly by grant n o 7 5 . 0 0 6 6 7

from the Consiglio Nazionale delle Ricerche and by a grant from

the Istituto Nazionale Ricerca e Cura Anziani (INRCA). F.Pedata

was the recepient of an INRCA research fellowship.

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