Nearochemistry International. Vol. 3, No. 6. pp. 411 to 416. 1981 0197-0186/81/060411-06502.00/1) Printed in Great Britain O 1981 Pergamon Press Lid

DOSE-RELATED EFFECT OF THE OXYTOCIN FRAGMENT (PROLYL-LEUCYL-GLYCINAMIDE) ON

• -MPT-INDUCED CATECHOLAMINE DISAPPEARANCE AND SEROTONIN LEVEL IN RAT BRAIN

GYULA SzAlg~,* GABOR L. KOV/,CS, L~OS BAL,/tSPIRIJf and G Y U L A TELEGDY

Institute of Pathophysiology and tlnstitute of Medical Chemistry, University Medical School, Szvged, Hungary

(Received 21 May 1981 ; revised 27 August 1981 ; accepted 15 September 1981)

Abstract--Graded doses of Pro-Leu-Gly-NH2 (3.5 × 10- 12 3.5 × 10- l l, 3.5 × 10-10 or 3.5 × 10 -9 tool) were administered into the lateral cerebral ventricle of rats. The noradrenaline level of the dorsal hippocampus was increased 30 rain after a dose of 3.5 × 10-10 tool Pro-Leu-Gly-NH2. The dopamine level was increased in the dorsal hippocampus and in the striatum. The serotonin level was increased in the hypothalamus, in the striatum and decreased in the dorsal hippocampus.

The catecholamine disappearance following 350mg/kg of a-methyl-p-tyrosine indicated an accelerated dopamine disappearance in the striatum for each dose studied, while the hypothalamic noradrenaline disap- pearance was inhibited by a dose of 3.5 × 10- i t tool of Pro-Leu-Gly-NH2.

The data indicate that Pro-Leu-Gly-NH2 induces dose and region-dependent changes in the cerebral mono- amine metabolism. The striatal dopamine and hypothalamic serotonin metabolism appeared to be the most sensitive for intraventricular Pro-Leu-Gly-NH2.

PLG has been shown to improve clinical symptoms of Parkinson's disease (Kastin and Barbeau, 1972; Gerstenbrand, Poewe, Aiehner and Kozma, 1979). In addition to this central nervous system effect, the tri- peptide also affects morphine tolerance and depen- dence (van Ree and de Wied, 1976; Bhargav& Walter and Ritzmann, 1980) and restores puromycin-induced (Fiexner, Fiexner, Hoffman and Walter, 1977; Flexner, Flexner, Walter and Hoffman, 1978) and electroconvulsive shock-induced amnesia (KovAcs, Rib/trszki, Nagy, Baldspiri and Telegdy, unpublished observations). However, the mode of action of PLG on the neuronal system is unclear as yet. Friedman, Friedman and Gershon (1973) reported an accelerated dopamine synthesis by PLG in tyrosine incorporation studies. Versteeg, Tanaka, de Kloet, van Ree and de Wied (1978) have shown that intraventricular admin-

* Correspondence to: Gyula Szab6, Institute of Patho- physiology, University Medical School, H-6701 Szeged, Semmelweis u. 1. P.O.B. 531, Hungary.

Abbreviations: PLG, Pro-Leu-Gly-NH2; prolyl-leucyl. glycinamide; NA, noradrenaline; DA, dopamine; 5-HT, serotonin; icy, intracerebroventricular; ,,-MPT, ~,-methyl- ~-tyrosine methyl ester HCl.

NC.L 3~6--~

istration of PLG induces region-specific effects in nor- adrenaline and dopamine turnover, including an ac- celeration of a-MPT-induced dopamine disappear- ance in the caudate nucleus. Schulz, Kov~cs and Telegdy (1979) investigated turning behaviour in sub- stantia nigra-lesioned rats and concluded that PLG affects the nigrostriatal dopaminergic system at a pre- synaptic level. However, the negative reports concern- ing the interaction of PLG with monoamine neuro- transmission are also numerous Kostrzewa, Kastin and Spirtes (1975) reported that PLG did not alter the dopamine turnover in the striatum. Neither have these authors (Kostrzewa, Fukushima, Harston, Perry, Fuller and Kastin, 1979) been able to replicate the studies of Versteeg et al. (1978); they instead sug- gested that PLG affects the striatal dopamine system at the postsynaptic site (Kostrzewa, Kastin and Sobrian, 1978). Their subsequent experiments, how- ever, seem to exclude a direct action of PLG on the postsynaptic receptors (Kostrzewa, Hardin, Snell, Kastin, Coy and Bymaster, 1979),

Since various doses of PLG have been used in the above studies and the effect of PLG on the central neurotransmission might depend on the dose and

411

412 GYULA SZABO et al.

route of administration, we decided to investigate the action of graded doses of PLG on the steady-state levels and on the :(-MPT-induced disappearance of NA and DA, as well as the steady-state level of 5-HT in various brain areas. For measurements of steady- state levels the animals were sacrificed 30 rain after an intracerebroventricular injection of the tripeptide. This interval was selected on the basis of our behav- ioral experiments, where the tripeptide was most effective within 1 h after treatment.

EXPERIMENTAL PROCEDURES

Male CFY rats weighing 150-200 g were kept under arti- ficial illumination (light between 6 a.m. and 6 p.m.). They received food and water ad libitura. The animals were anaesthetized with sodium pentobarbital (Nembutal @, 40 mg/kg) and a polyethylene cannula was inserted into the right lateral cerebral ventricle. The cannula was fixed to the skull by screws and dental acrylate. After a recovery period of 7 days. free.moving animals received an intracer- ebroventricular injection of PLG in doses of 3.5 x 10-1:, 3.5 x 10 -11, 3.5 x l0 - t ° or 3.5 x 10-9 tool, dissolved in 2 ~l of physiological saline. For the estimation of the steady-state levels of NA, DA and 5-HT, the animals were decapitated 30 rain after the treatment. A separate group of rats was injected with PLG {3.5 x 10- ' :-3.5 x 10 -1° too!) for the measurement of catecholamine disappearance after ~-MPT administration. In this latter group, icy injection of the peptide was given 30rain after pretreatment with :t-MPT (350 mg/kg i,p.). The rats were decapitated 3 h after the peptide treatment. Control groups received the vehicle. The brains were removed and dissected into the following regions: hypothalamus, mesencephalon, striatum, and dor- sal hippocampus. Brain regions were assayed for mono- amines by the fluorimetric mzthod of Jacobowitz and Richardson {1978) as sfightty modified by Szab6, Kovfics and Telegdy (unpublished observations). Briefly, brain samples were homogenized in 5 ml of ice-cold n-butanol containing 0.72 ml 0.01 N HCI. After centrifugation. 3 ml of the butanol supernatant were added to 3.0 ml of 0.1 M phosphate buffer (pH 7.0). After vigorous mixing and cen- trifngation the organic supernatant was discarded and 2.8 ml of the inorganic phase wer~ oxidised by iodine. NA was measured on 395 nm exciting and 475 nm analysing wavelength, DA was estimated on 325 nm exciting and 380 nm analysing wavelength. For the 5-HT measurements. 2 ml of the butanol extract were added to 4 ml of n-heptane and 3 ml of 0.05 M phosphate buffer. After mixing and centrifugation the phosphate phase was supplemented with 0.1 M ninhydrine and 5-HT was measured on 365nm exciting and 490 nm analysing wavelength. The results are expressed as nmol transmitter/g tissue. Statistical analysis was performed by Anova, followed by Student's t-test. A probability level of 0.05 was accepted as a significant differ- ¢ n c e .

Materials

PLG was obtained from Richter, Budapest and ~t- methyl-p-tyrosine-methyl ester HC1 from Sigma. St. Louis. Other chemicals were of analytical grade from various commercial sources.

RESULTS

PLG and steady-state lerel of monoamines

PLG increased the steady-state levels of NA and DA in the dorsal hippocampus (3.5 x 10- lo tool), as well as the DA content of the striatum ('3.5 × 10-12 and 3.5 x 10-1~ mol l There was no change in the steady-state level of either catecholamine in the hypo- thalamus and mesencephalon.

PLG increased the steady-state level of 5-HT in the hypothalamus t3.5 x 10 -~2 and 3.5 × 10 - t l mol) and in the striatum (3.5 >< 10-1~, 3.5 × 10-1° and 3.5 × 10 -9 tool). On the other hand, there was a de- crease in the 5-HT content of the dorsal hippocampus (3.5 × 10 -11 tool) (Table 1).

PLG and catecholamine disappearance after ~-MPT administration

Data on the ~(-MPT-induced catecholamine disap- pearance are summarized in Table 2. ~t-MPT treat- ment decreased the NA and DA levels in each brain region significantly, except for the DA level in the mesencephalon, where the decrease was modest. In the :t-MPT-treated animals. PLG prevented the NA disappearance in the hypothalamus (3.5 x 10 -~t mol). Striatal DA ~ p p e a r a n c e was significantly in- creased by each dose of PLG (Table 2).

DISCUSSION

The present experiments indicate that icy treatment with PLG affects the steady-state levels and the x-MPT-induced disappearance of brain catechoi- amines, as well as the steady-state level of serotonin. This effect is clearly dependent on the dose of the tripeptide and also on the brain regions sampled. In accordance with the data of Versteeg et al. {1978), it has been found that PLG increased the ~-MPT- induced DA disappearance in the striatum, which is also in support of the reports of Kastin and Barbeau {1972) and Gerstenbrand et al. {1979), who found potentiating effects of PLG on L-DOPA therapy in Parkinsonian patients. In addition, PLG increased the steady-state level of DA and 5-HT in the striatum. Interestingly, no effect on DA occurred with the high- est dose of the peptide.

It is worthwhile to mention that PLG increased the catecholamine levels in the dorsal hippocampus. which structure has an outstanding position in memory and learning processes {Kimble, 1963). Since the NA metabolism in the dorsal hippocampus is cor- related with memory processes (Kovacs, Versteeg, de Kloet and Bohus, 1981) it cannot be excluded that the

Tab

le 1

. Dos

e-re

lale

d ef

fect

of

PL

G o

n br

ain

mon

oam

ine

leve

ls

3.5

x 10

-tZ

mol

3.

5 x

!0-1

1 m

ol

3.5

x IO

-n°m

ol

3.5

x lO

-9.m

ol

Bra

in r

egio

n/m

onoa

min

e C

ontr

ol

PL

G

PL

G

PL

G

PL

G

Hyp

otha

larn

us

Nor

adre

nali

ne

8.98

± 0

.30*

(20)

8.

37 +

0.4

2 {7

) 9.

39 ±

0.7

0(8)

9.

57 +

0.3

008)

8.

81 ±

0.3

5 (7

) D

opar

nine

9.

73 ±

0.7

2(20

) 12

.94

± 2,

66(6

) 10

.05

± 1.

64(8

) 10

.31

± 0.

72(1

9)

10.9

2 ±

1.10

(8)

Sero

toni

n 9.

77 +

0.5

6(18

) 13

.19

± 1.

34:[

(8)

12.8

1 ±

!.54

t (7)

9.

32 ±

0.5

1 (1

5)

10.9

7 ±

0.95

(4)

Mes

ence

phal

on

Nor

adre

nali

ne

2.90

± 0

.12

(19)

2.

62 ±

0.2

7 (8

) 2.

78 ±

0.2

1 (7

) 2.

83 ±

0.1

5 (2

1)

2.62

± 0

.22

(8)

Dop

amin

e 6.

80 ±

0,3

1 (2

1)

6.19

± 0

.63

(8)

6.59

+

1.09

(7)

6.46

± 0

.39(

20)

7.25

± 0

.20(

7)

Sero

toni

n 4.

79 ±

0.3

6(17

) 5.

89 ±

0,5

3(8)

5.

68 ±

0.3

9(7)

3.

98 +

_ 0.3

4(16

) 6.

30 ±

0.9

0(4)

Dor

sal

hipp

ocam

pus

Nor

adre

nali

ne

1.00

± 0

.06

(21)

1.

24 +

_ 0.1

2 (6

) 0,

86 +

0.1

3 (8

) 1.

30 ±

0.0

6§(1

9)

1.08

± 0

.07

(8)

Dop

arni

ne

3.45

± 0

.18

(21)

3.

09 ±

0.2

0 (6

) 3.

52 ±

0.2

5 (7

) 4.

63 ±

0.2

8§ (2

0)

4.32

± 0

.67

(7)

Sero

toni

n 4.

53 ±

0.3

2 (1

8)

4.14

± 0

.56(

8)

3.25

± 0

.28t

(7)

3.80

_+

0.30

(16)

3.

81 ±

0.3

1 [4

)

Stri

atum

D

opam

ine

46.5

0 +

1.76

(22)

62

.99

_+ 1

.701

1 (7)

57

.87

+ 1.

95§(

7)

48.3

1 +

2.03

(18)

41

.81

± 2.

55 (8

) Se

roto

nin

3.38

± 0

.15(

18)

4.18

± 0

.55(

8)

5.51

± 0

.441

1 (8)

4.

43 _

+ 0.

27§(

17)

4.40

+ 0

.51:

~ (4

)

* M

ean

+ S.

E.M

. (n

mol

/g).

tP

<0.

05.

:[ P

< 0.

02.

§ P

< 0.

01.

II P

< 0.

001.

P

LG

was

adm

inis

tere

d in

to t

he l

ater

al c

ereb

ral

vent

ricl

e an

d th

e an

imal

s w

ere

deca

pita

ted

30 rn

in a

fter

tre

atm

ent.

4~

Tab

le 2

. D

ose-

rela

ted

effe

cl o

f P

LG

on

a-M

PT

-ind

uced

cat

echo

lam

ine

disa

ppea

ranc

e

3.5

x 10

-12m

ol

3.5

x 10

-11t

ool

3.5

x 10

l°

mo

l B

rain

reg

ion/

Mon

oam

ine

Sal

ine

+ sa

line

S

alin

e +

a-M

PT

P

LG

+

a-M

PT

P

LG

+ a

-MP

T

PL

G

~- a

-MP

T

Hyp

otha

lam

us

Nor

adre

nali

ne

13.7

1 ±

0,53

"*(1

5)

8.53

+ 0

.47:

[ (1

5)

9,46

or 0

.83(

8)

11.3

5 +

0.61

§(8)

8.

03 i

0,

58(8

) D

opam

in¢

11.2

6 +

1.24

(14)

5.

84 _

+ 0.

89t(

12)

4.60

+ 0

.46(

5)

7.77

or

1.40

(8)

5.55

°r

1.90

(5)

Mes

ence

phal

on

Nor

adre

nali

ne

3.67

or

0.17

(14

) 2.

06 ±

O. 1

0~ (1

5)

2.45

± 0

.23

(8)

2.12

± 0

.11

(7)

2.21

or

0.16

(8)

Dop

amin

¢ 9.

20 o

r 0.

72(1

5)

8.02

or

0.86

(13)

7.

43 o

r 0.6

9(8)

8.

23 ±

1.

07(7

) 8.

25 o

r 0.

48(7

)

Dor

sal

hipp

ocam

pus

Nor

adre

nali

ne

1.29

± 0

.08(

15)

0.53

± 0

.08:

[(13

) 0.

49:1

: 0.

05(8

) 0.

47 ±

0.0

6(8)

0.

46 +

0.0

7(8)

D

opam

in¢

7.96

or 0

.94(

14)

5.18

or 0

.67*

(1 I)

4.

84 o

r 0.5

3 (7

) 6.

01 o

r 0.6

7 (8

) 5.

70 o

r 0.6

5 (8

)

Str

iatu

m

Ek~

pam

ine

74.2

2 i

2.87

(14

) 36

.78

or i

.48~

, (15

) 28

.89

± 1.

66tl

(7)

31.4

5 or

2.2

0§(8

) 29

.24

or 0

.74¶

(7)

* P

< 0.

05 v

s sa

line

con

trol

. t

P <

0.01

vs

sali

ne c

ontr

ol.

P <

0.00

1 vs

sal

ine

cont

rol,

§

P <

0.05

vs

:-M

PT

con

trol

. IJ

P <

0.01

vs

a-M

PT

con

trol

. ¶

P <

0.00

1 vs

a-M

PT

con

trol

. **

Mea

n i

S.E

.M (

nmol

/g).

A

nim

als

wer

e tr

eate

d by

a-M

PT

(35

0 m

g/kg

, i.p

.~ f

ollo

wed

30

rain

lat

er b

y an

icy

inj

ecti

on o

f P

LG

or

sali

ne.

Rat

s w

ere

sacr

ific

ed 3

haf

tcr

the

icy

inje

ctio

n,

PLG and brain monoamines 415

hippocampal effects of PLG are coupled with the anti-amnesic action of the tripeptide (Fiexner et al., 1977; Kov~ics et al., unpublished observations).

The tripeptide inhibited the u-MPT-induced NA disappearance and increased the 5-HT level in the hypothalamus. The possibility might be worth con- sidering as to whether the hypothalamic effects of the tripeptide are related to the reported inhibition of se- cretion of melanocyte-stimulating hormone (Cells, Taleisnik and Walter, 1971, Nair, Kastin and Schally, 1971).

The effects of the tripeptide on striatal DA metab- olism are in agreement with similar findings of others (Versteeg et al., 1978) and provide a likely neuroana- tomical background for the beneficial effect of PLG in Parkinson's disease (Kastin and Barbeau, 1972; Ger- stenbrand et al., 1979).

In conclusion, the data indicate that PLG exerts dose-related effects on the cerebral catecholamine and indolamine metabolisms; however, these effects are not necessarily parallel with the injected amount of the tripeptide. In some instances, U-shaped and inverted U-shaped dose-response curves were ob- served. The reason for these effects is not clear as yet. It should be emphasized, however, that the tripeptide affects the central nervous system in a very complex manner, involving prcsynaptic (Schuiz et al., 1979), post-synaptic (Kostrzewa et al., 1978) neuronal ele- ments, opiate receptors. (Kastin, Nissen, Zadina, Schally and Ehrensing, 1980) and cyclic nucleotides (Spirtes, Christensen, Harston and Kastin, 1978) etc. These factors may all have an impact on the influence of the peptide on neurotransmission and need to be further elucidated. It might be supposed that a pro- portion of the apparent contradictions in the litera- ture concerning the effect of PLG on neurotransmis- sion may be due to the widely differing doses of PLG used in different laboratories, although it should be noted that Versteeg et al. (1978) and Kostrzewa et al. (1979) used exactly the same dose and yet did not find similar changes. This apparent contradiction, together with the complex close and region-dependent changes found in this study, argue for the necessity of further neurochemical experiments to analyse the mode of action of PLG on the central nervous system.

Acknowledgements--We are grateful to Mrs K. Kov~ics and Miss A. Szoke for skillful technical assistance.

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416 GYULA SZ,~6 et al.

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