Turnover of dopamine and norepinephrine in hypothalamus of normal and hypophysectomized rats

Pharmacological

Research Communications,

311

Vol. 4, No. 4. 1972

TURNOVEROF DOPAMINE AND NOREPINEPHRINE IN HYPOTHALAMUSOF NORMAL AND HYPOPHYSECTOMIZEDRATS and R. Nallar'

Ana M. Biscardi'

Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, CQtedra de Fisiologia Humana, Buenos Aires (Argentina). Received

28 August

1972

SUMMARY The concentration catecholamines

(dopamine,

term hypophysectomized increases

does not

It

norepinephrine) gland

accepted

play

mainly

of the pituitary Several

show that

process

is no agreement

of the

catecholamine it

states,

increases

in castrated

that

the brain

(Everest,

catecholamines rol

as

is generally

control

1964;

Coppola,

and turnover

(Anton-Tay

1968).

authors

accepted

1966;

1968).

catecholamines

among the different

rats

increase

and Langemann,

and Danhof,

concentration

endocrine

this

the hypothalamic

(Lichtensteiger

1966; Kamberi

papers

but

an important

into

Fuxe and Hokfelt,

there

and the turnover

values.

is generally

neurotransmitters,

in long-

hormone to such

the concentration

the normal

INTRODUCTION

the ovulation

decrease

dopamine and norepinephrine,

reach

(dopamine,

norepinephrine) of luteinizing

both:

of hypothalamic,

of hypothalamic

rats.

The administration animals

and turnover

that

control Although

in respect in various their

and Wurtman,

turnover 1968).

Consejo National de Investigacio0 Established Investigator, nes Cientificas y Tecnicas (National Research Council) of Argentina.

312

Pharmacological

Anton-Tay the

and Wurtman (1969)

same effect

as castration.

hypophyaectomy

does not alter

norepinephrine

in rata.

demoatrated

that

methylester

(H &t/68),

hydroxylase,

for

METHODS

hypophysectomized Smith

(1927,

constant

with

with use,

and turnover

were kept

hypophysectomy,

of sixty

and injected

group were injected

animals.

o Obtained

by

in a room at a 12 hours soluble

of daily chow,

terramicine

with

the

jugular

the rata

One of these

subcutaneously

with

were divided groups

ovine

3 times a day, of IJH, forty

were

luteinizing

for 10 days. animals of each

dl-3,4-dihydroxyphenylalanine-H3

The administered into

described

same age were used as control.

hormone (NIH-IX-S9) 5 mcg/rat, The day after the last injection

injected

g were

ad libitum.

Two months after

(dopa-H3)'.

of

Forramez laboratory

raw meat and water

and

are concerned.

200-250

of the

in two groups

1971).

hormone is

to the technique

They were fed with

in

et al.,

luteinizing

Normal male rats

castrated

of inhibition

of hypophyaectomy

of 25 0 C and with

temperature

supplemented animal

or

(Ahren

weighing

The rata

1930).

illumination. for

castrated

concentration

according

has been

of the tyrosine-

catecholamines

male rats

of tritiated

of alpha-methyl-tyroaine-

dopamine and norepinephrine Adult

it

same extent

treatment

what the

hypothalamic

the

has

that

turnover

hand,

paper the effects

substitutive

studied

other

in normal,

of brain

In the present of the

the brain

Vol. 4, No. 4, 1972

hypophyaectomy

They show also

On the

animals

Communications,

that

an inhibitor

produces

synthesis

suggest

the administration

hypophysectomized the

Research

dose was 15OpCi/lOO vein

from the New England

in 0.5 ml of saline. Nuclear

Corporation

g b.w. The

Pharmacological

animals

Research Communications,

were killed

anesthesia

2.5,

dopa-H3.

glands

residuals

after

and homogeneized homogenizer. minutes

death

in ice-cold

The homogenates

and were then

4O C (Bertler

injection

for

of

possible

of a dissecting

were inspected. or trophic

residuals The rats

aspect

of target

et al.,

the hypothalamus

were removed

0.4 N perchloric

acid

were allowed

centrifuged 1958).

an equal

the

were combined.

supernatants In order

at 900 g for

volume

catechol-acids,

the

extracts

chromatographed

on CG-50 exchange

(1957).

The 1 N acetic

and rechromatographed

eluates

oxide

(Dowex 50, H+, 6 by 60 mm, wet). and norepinephrine the column.

was eluted

was eluted

hydrochloric

(1958).

The

on AG-50 WX~ resin

A carrier

amount of dopamine and passed through

washed with

distilled

water. after

dopamine in 2.5 N hydrochloric

acid.

Each

was rechromatographed

eluate

was used for

on aluminium

and Waldeck

and Lishajko

oxide

counting.

of dopamine was carried

the method of Carlsson by Euler

with

was

acid,

The determination described

1968).

1 N hydrochloric

eluate

and an aliquot

e!t al.

was added to the eluate

The column was then

Norepinephrine

at pH 8.4

(Crout,

was chromatographed

labeled

and Goodall

of dopamine from norepinephrine

eluate

and

Amberlite,

were adjusted

out by the method of Bertler

aluminium

acid

at pH 6.1 and

(IRC-50,

oxide

at

was

dopa-H3 and the

to Kirshner

on aluminium

The separation carried

acid

precipitate

resin

30

20 minutes

were adjusted

according

Na+, 6 by 100 mm, wet),

for

of 0.4 N perchloric

to remove the residual acid

in a glass

to stand

The protein

washed once with

with

the

ether

were discarded. Inmediately

which

light

after

was examined

Also the target

showed pituitary

organs

under

5, 10 and 20 hours turcica

313

by the naked eye and by the aid

microscope. which

by decapitation

The sella

of pituitary

Vol. 4, No. 4, 1972

(1961)

(1958).

out according The method

was used for

the

314

Pharmacological

determination activity

of norepinephrine.

of different

Automatic

Liquid

of a solution

containing

standards

per minute

standard

error

differences

for

activity

as count

in a Packard

Fourteen

milliliters 10 g of 2,5-

and Lyman, 1967).

were corrected

The specific

of radio-

was used as scintillation

the background

and 30 cpm. Internal

Vol. 4. No. 4. 1972

g of 1,4-bis-[2-(5-phenyloxazolyl))

(Draskoczy

were used;

counts

Counter.

of dioxane

the eluates

the

was performed

100 g of naphthalene,

and 0.25

benzene per liter bottles

samples

Communications,

The determination

Scintillation

diphenyloxazole for

Research

counts

Plastic

counting

ranged between

were added to all

25

samples

and

quenching.

of each catecholamine

per millimicromole.

was expressed

The values

of the media and the

were calculated

medium

significance

according

with

for

the

of the

Snedecor

(1956).

RESULTS A) Dopamine and norepinephrine of normal, castrated

concentration

hypophysectomized rats

treated

and norepinephrine Hypophysectomy

mentioned both

the values

On the

the normal

values

B) Conversion

other

(figure

with

decrease

in the not

hormone as concentration

significant

of when

hypophysectomized

such an increase

does not reach

1).

in hypothalamus

We have used a radioactive

in

and norepinephrine.

luteinizing

of untreated

of dopa-H3 to labeled

norepinephrine

studied.

dopsmine

is however

hand,

of dopamine

a significant

an increase

which

catecholamines,

animals.

animals

induces

of rats

with

of hypothalamic

above,

hormone.

in the concentration

was associated of these

compared with

luteinizing

among the groups

the concentration The treatment

and hypophysectomized-

with

There were differences

in hypothalamus

dopamine and of normal

rats.

amine precursor

for

studing

Pharmacological

Research Communications,

0.8

Vol. 4, NO. 4, 1972

315

.

0.6 g g .CI

0*4

g

0.2

0”

0.1 -

-z-

00 i 0.3

1I 2

3

%

-

2

3

Figure 1: Dopamine (DA) and norepinephrine (NE) concentration in the hypothalamus of: 1) normal rats, 2) hypophysectomized rats and 3) hypophysectomized-castrated rats treated with LH. the turnover reported

of catecholamines

that

the results

obtained

measured by means of labeled those

found

"Jlowinsky,

with

Neff

et al.,

The administered dopa-IS3 only found the

0.8-1.0

starting

dopamine

point

for

acid

chromatographed effluent

to

and

after

the

from the

injection

of

radioactivity that

time

the turnover

was

was taken

as

of labeled

and norepinephrine.

To measure the perchloric

studing

(Iversen

disappeared

injected

therefore

in the hypothalamus,

rates

1969). hours

$ of the

has been

are similar

inhibition

dopa-H3 rapidly

Two and a half

It

in the turnover

amine precursors

amine synthesis

1966;

hypothalamus.

in hypothalamus.

of this

amount of the residual

extracts

of hypothalamus

on

resin

CG-50

resin,

dopa-H3,

the

were

to remove catecholamines.

containing

dopa-H5 and other

The

316

Pharmacological

Research Communications,

Vol. 4, No. 4, 1972

DA

\

2.5

I 20

I 10

L 5

100

HO0 rs

Figure 2: Rate of radioactivity fall in hypothalamic dopamine (DA) and norepinephrine (NE). The abscissa indicates the time after the injection of tritiated dopa and the ordinate the counts per minute per hypothalamus. derivatives

was then

chromatographed

remove the non-catechol was counted

(Udenfriend

authors

and Zaltzman-Nirenberg,

Draskoczy,

1964;

Draskoczy

The radioactivity that

This

event

counter. in rats

faster

with

and in

the results

other

1963;

as it

the turnover

than

that

eluate

animal

Burack

species

and

and Lyman, 1967).

due to norepinephrine,

dopamine is

to

Such a rapid

due to dopamine decreases

shows that

oxide

and the acid

of dopa-H3 was in accord

by other

than

derivatives,

in a scintillation

disappeareance obtained

acid

on aluminium

rate

is

more rapidly

shown in figure

of hypothalamic

one of norepinephrine.

2.

Pharmacological

Research Communications,

Vol. 4. No, 4, 1972

317

Hours

Figure 3: Turnover of dopamine (DA) and norepinenhrine (ME) in the hypothalamus of: 1) normal rats, 2) hypophysectomized rats treated with LH. rats and 3) hypophysectomized-castrated C) Turnover

of labeled

hypothalamus

dopamine and norepinephrine

of normal,

hypophysectomized

hypophysectomized-castrated

rats

treated

in

and with

luteinizing

hormone. Our results labeled is

clearly

significantly

decreased

increases

the turnover

the untreated In order

rate

but

not

of hypothalamic

among the

three

in comparison

groups

with

of hypothalamic

in a significant numerically

the normal

of rats rats.

dopamine and extent

animals

when compared

(figure

the difference

dopamine and norepinephrine of rats,

rate

hormone in hypophysectomized

hypophysectomized

to express

the turnover

in hypophysectomized

of luteinizing

norepinephrine, with

that

dopamine and norepinephrine

The administration rats

indicate

the percentage

3).

in the turnovers

change of

318

Pharmacological

Research Communications,

Table I: Percentage decrease of specific (DA) and norepinephrine (NE) in 20 hours of: 1) normal rats, 2) hypophysectomized hypophysectomized-castrated rats treated

Vol. 4, No. 4, 7972

activity of dope,mine in the hypothalamus rats and 3) with LH.

2

55 + 4 (20)

45 + 4 (18)

3

68 + 3 (20)

59 + 4 (20)

O significant difference between normal and hypophysectomized rats and normal and hypophysectomized-castrated rats -treated with LH: P
activity

per 20 hours The results

catecholamine.

These results cntecholamines the partial treated

luteinizing

experimental

conditions decrease

administretion modify

this It

under

in the concentration

and norepinephrine.

of

rats

and rats

the adopted animal

and turnover

Moreover,

hormone does not

shows a of

the significantly

parameter. known that

such modifications the biochemical extents

show that

the hypophysectomized

modifications

the normal

in the turnover

of hypophysectomized

of luteinizing

is well

metabolic

I.

hormone.

Our experiments

dopsmine

each

in the hypophysectomized-castrated

DISCUSSION

labeled

the decrease

in hypothalamus

with

for

are shown in table

demostrate

recovery

significant

was determined

state.

practically

hypophysectomy

in different

are mainly is

also

to all

animal

the tissues

and that

by a decrease

processes

known that

widespread

species,

characterized

and physiological It

induces

which

these

in

subserve

modifications

of the body,

and that

Pharmacological

the

Research Communications,

tissues

most.

with

This

possible after

Vol. 4, No. 4. 1972

the highest

also

applies

to observe

metabolic

to the

a decrease

hypophysectomy, ionic

phosphatidic

and carbohydrate

is

in the three

now clearly

animal

The pituitary

that

factors:

substitutive

it

activity potentials,

etc. the

energetic

conditions

takes

by a general recovered

and nutritive.

general

that

control

depends upon

endocrine

fault

and specific

place

after

hypometabolic

castration.

of the entire

state

even by a total

metabolic

is much more intense

The lack than

activity

than

that

of balance

in the

of all

the pituitary

by supression

more important

the

lack

of action

following

one produced

control

Therefore luteinizing

we consider

that

the

hormones is much

of gonadal

administration

hormone to hypophysectomized recover

whose biochemical significant

certain

hypothalamic

manifestations

tendence

to normalize

of hypothalamic

animals, conditions,

steroids

the concentration

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partially

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in which

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can be only

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in a balanced

effects.The

suffer

of bioelectric

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tissues

fundamental

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a diminished It

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