Adrenal cortex — A newly recognized peripheral site of action of enkephalins

Vol. 97, No. 4, 1980

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December 31, 1980

Pages 1346-1353

ADRENAL CORTEX - A NEWLY RECOGNIZED PERIPHERAL SITE OF ACTION OF ENKEPHALINS K. R~cz, E. Gl~z, R. Kiss, Gy. Lada, I. Varga, K. Di Gleria**, K. Medzihradszky**, K. Lichtwald* and P. Vecsei*

S. Vida,

2nd Department of Medicine, Semmelweis University, School of Medicine, H-I088 Budapest, Hungary *Department of Pharmacology, University of Heidelberg, Germany and **Central Research Institute of Chemistry, Hungarian Academy of Sciences, Budapest, Hungary

Received October 27, 1980 SUMMARY

Two naturally occuring enkephalins, Leu- and Met-enkephalin, were shown to inhibit adrenal corticosteroid biosynthesis. In isolated rat adrenal cell system corticosterone, desoxycorticosterone and aldosterone, and in isolated bovine adrenal cells cortisol, corticosterone and aldosterone production were markedly depressed in a dose-dependent fashion, both under basal conditions and following stimulation with ACTHI_24. The inhibitory action of enkephalins on corticosteroid synthesis could be augmented by increasing the Ca2+-concentration in the cell-containing medium. The demonstration that opioid peptides interfere with corticosteroidogenesis directly at the adrenal level points to the existence of a peripheral neuroendocrine relationship.

INTRODUCTION The physiological role and biological importance of enkephalins has become subject to growing interest,

and recent data on

this peptid group are quite intriguing. Besides numerous non-hormonal effects, Met- and Leu-enkephalin as well as their recently synthetized analogues have been shown to interfere with endocrine functions, of which obviously the hypophysis,

the organ with the

closest link to the central nervous system, has received the greatest attention. Thus it was found that the Met-enk@phalin analogue [D-AIa 2, MePhe 4, Met(O)-enkephalin-ol

(DA~IME~ decreased plasma

ACTH and cortisol levels in healthy individuals

0006-291 X/801241346-08501.00/0 Copyright © 1980 by Academtc Press, Inc. All rights o f reproduction in any form reserved.

1346

(i), in patients

Vol. 97, No. 4, 1980

with Addison

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and C u s h i n ~ s

could be o b s e r v e d cortisol

levels

tion to this system,

centrally

which

phalin-like various

over

had b e e n

circulatory

cytoma

mediated

(4),

renal m e d u l l a structures,

recent

medulla

mone which

plasma

with

effect

was

was

ACTH

(3).

and

In addi-

blood

connections,

cortex.

Leu-enkeof

(5) and p h a e o c h r o m o -

of the dog

of dogs

as part

in the adre-

in the adrenals

medulla

(7), m o r e -

(8). A l t h o u g h

the ad-

of the p e r i p h e r a l

nervous

(9) h a v e

and L e u - e n k e p h a l i n

detected

anatomical

the adrenal

effluent

effect

on the h y p o p h y s e o - a d r e n a l

rich

adrenal

observations

is d i f f e r e n t

vasopressin

demonstrated

be r e g a r d e d

is i n d e p e n d e n t

whose

enkephalin

and venous

venous

Met-

subjects

with

in h u m a n

in arterial

might

inhibitory

to have

immunoreactivity

(5, 6),

synthesis

that

and nervous,

species

(2), and this

stimulated

is k n o w n

in the adrenal

adrenal

in normal

it is of i n t e r e s t

nal m e d u l l a both

also

disease

shown

that

in the bovine

are d e r i v e d

of b e t a - e n d o r p h i n e ,

f r o m that p r e v a i l i n g

thus

from a p r o h o r the p a t h w a y

in the central

of

nervous

system. In v i e w of these peptids

may

act not o n l y

but p o s s i b l y

mediate

investigating exerted rect

effect

whether

responses

enkephalin,

on the target action

in vitro,

using

the e n d o g e n o u s

and p i t u i t a r y To test

of the two n a t u r a l l y was

studied

to in vivo

adrenal

it seemed w o r t h

to its central levels,

effects

has any di-

this p o s s i b i l i t y ,

occurring

the

pentapeptides,

on c o r t i c o s t e r o i d cell

opioid

and n e u r o m o d u l a t o r s

as well,

in a d d i t i o n

organs.

the i s o l a t e d

comparably

that

as n e u r o t r a n s m i t t e r s

hormonal

and M e t - e n k e p h a l i n

respond

indicating

at the s u p r a h y p o p h y s e a l

peripheral Leu-

data

system which

biosynthesis is known

to

impacts.

~TERIALS

AND METHODS

A d r e n a l s were o b t a i n e d from m a l e W i s t a r rats w e i g h i n g 300 to 400 g; until d e c a p i t a t i o n , the animals w e r e a l l o w e d free access to

1347

Vol. 97, No. 4, 1980

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food and water. B o v i n e adrenals w e r e p u r c h a s e d as w h o l e , f r e s h l y rem o v e d glands from a s l a u g h t e r - h o u s e ; the glands were k e p t until use in K r e b s - R i n g e r b i c a r b o n a t e b u f f e r at 4 C ° . I s o l a t i o n of adrenal cells from the q u a r t e r e d rat a d r e n a l s and slices of the b o v i n e adrenal c o r t e x was u n d e r t a k e n similarly, by d i g e s t i o n w i t h C o l l a g e nase, T y p e I, and D N A - a s e I (Sigma C h e m i c a l Co., Ltd.) and m e c h a nical d i s p e r s i o n using a p l a s t i c p i p e t t e (i0). The cell suspensions c o n t a i n e d 8 x 105 or 1.2 x 106 cells in a final v o l u m e n of 0.6 ml K r e b s - R i n g e r b i c a r b o n a t e b u f f e r w i t h glucose, 0.2 %, b o v i n e serum albumin, 0.5 %, Na +, 143.4 mmol/l, K +, 5.9 mmol/l, Ca 2+, 2.54 or 7.62 mmol/l, at pH 7.4. All t e s t e d substances, viz. A C T H I _ 2 4 (Synacthen, Ciba Ltd., Basel), M e t - e n k e p h a l i n (molecular weight; 574), and L e u - e n k e p h a l i n (molecular weight; 556), b o t h s y n t h e t i z e d by M e d z i h r a d s z k y (ii), w e r e added to the cell s u s p e n s i o n s prior to incubation, and all tested c o n c e n t r a t i o n s w e r e studied in 5 parallel sets of i n c u b a t i o n and in 4 d i f f e r e n t series of experiments. The s u s p e n s i o n s w e r e i n c u b a t e d at 37 C ° in a m o v i n g w a t e r b a t h in an a t m o s p h e r e of 95 % 02 and 5 % CO 2 for 120 min; the m e d i a w e r e t h e n frozen to -20 C ° , and t h a w e d and c e n t r i f u g e d for steroid det e r m i n a t i o n s . C o r t i s o l (F), c o r t i c o s t e r o n e (B), d e s o x y c o r t i c o s t e r o ne (DOC), and a l d o s t e r o n e (Aldo) c o n c e n t r a t i o n s w e r e m e a s u r e d w i t h direct radioimmunoassay, using immune sera of h i g h specific a c t i v i ty (12, 13, 14). The s e n s i t i v i t y of the assays was 30 pg for F, 25 pg for B, 20 pg for DOC, 5 pg for Aldo. The i n t r a a s s a y and interassay c o e f f i c i e n t s of v a r i a t i o n w e r e 6 % and i0 %, respectively. Student's t-test was used for s t a t i s t i c a l e v a l u a t i o n of the data.

RESULTS In the rat and b o v i n e kephalins

exerted

ticosteroid ship

an e s s e n t i a l l y

biosynthesis.

in both cell

1 demonstrates

the effect

sence of 7.62 m m o l / l

tion of 48 % and tion e m p l o y e d

similar

inhibitory

41%,

significantly.

marked

in the isolated

adrenal

occurring

with

concentrations

higher.

1348

effect

causing

the rate of A l d o

system,

Figure

in the prewas

the m a x i a reduc-

even the lowest

of the synthesis

cell

on correlation-

w i t h DOC,

(10 -4 mol/l)

decreasing

Inhibition

cells

The b l o c k i n g

as p r o n o u n c e d

respectively,and

(lO -I0 mol/l)

action

en-

i0 -IO - 10 -4 mol/l,

by rat adrenal

and almost

tested

four c o r t i c o s t e r o i d s .

of M e t - e n k e p h a l i n ,

synthesis

both

a clear d o s e - r e s p o n s e

and for all

of M e t - e n k e p h a l i n

thesis

cant d e p r e s s i o n

suspensions

of Ca 2+ in the medium.

with Aldo,

mum concentration

cell

There was

systems

on the rate of steroid

most marked

adrenal

concentraand DOC syn-

of B was

statistically

less signifi-

of 10 -7 mol/l

and

Vol. 97, No. 4, 1980

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i~'0 1(39

100° 4/ ,

1(37

'

105 164 r.o~ M,t-

l

9O z o

8o o oc {3.

_

70 %

on,,

60

o i.--

g u

50

Fig. 1.: Met-enkephalin decreased, in a dose-dependent manner, the rate of aldosterone (~-4~), desoxycorticosterone (V If'), and corticosterone (~-43) synthesis in isolated rat adrenal cells incubated in the presence of 7.62 mmol/l Ca 2+. The 50 % of maximum inhibition was attained for Aldo by 1.3 x iO -I0 mol/l Met-enkephalin, for DOC by 2.2 x i0 -I0 mol/l, and for B by 9.5 x iO -IO mol/l. Degree of significance: * p < 0 . O 5 ; * * p ~ O . O l ; * * * p ~ O . O O l ; n.s. = not significant. Baseline values (mean + S.D.) were as follows (in pg/l.2 x 106 cells/120 min)~ Aldo, 913 ~ 158; DOC 8716 + 652; B, 39880 + 2875.

Considering synthesis bovine

(15),

adrenal

the cell

Met-enkephalin, bovine

adrenal

in the m e d i u m . marked cited

the

to t h e

in t h e

species

isolated

7.62 m m o l / l

of

there was production

B and,

bovine

in

of Ca 2+

Met-enkephalin,

mainly

Aldo was most markedly

also

in t h e r a t a n d

characteristics,

systems:

steroid

the e f f e c t s

biosynthesis of

of

studied

I summarizes

inhibited by

was

of corticosteroid

two c e l l

10 -5 m o l / l , synthesis

in t h e p a t h w a y

in t h e p r e s e n c e

changes

in t h e

adrenal

Table

on steroid

Owing

F and DOC

as in t h e r a t

suspension.

systems,

of

differences

of M e t - e n k e p h a l i n

cell

by enkephalin

extent,

effect

10 -5 m o l / l ,

difference

concentration

species

a eli-

in a

to a l e s s e r

adrenal

depressed

cells,

where

by this

compound. The well substances

documented

specifically

dependence stimulating

1349

o n Ca 2+ c o n c e n t r a t i o n corticosteroid

of t h e

biosynthesis

VOI. 97, No. 4, 1980

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table COMPARISON

OF THE

STEROID

INHIBITORY

SYNTHESIS

E F F E C T OF M E T - E N K E P H A L I N

605 + 59

DOC

3032 + 661

B

cells

Met-enkephalin 10-5 m o l / l

Control

Bovine

-

adrenocortical

69 + ii

-

cells

Met-enkephalin 10 -5 m o l / l

Control

265 + 27*** 1910 + 220**

16090 + 1690 11400 +1270***

F

ON C O R T I C O -

OF RAT A N D B O V I N E A D R E N A L C E L L S

Rat a d r e n o c o r t i c a l

Aldo

I

63 + 9 n's"

2750 + 233

1580 + 280*

3060 + 261

1252 + 487***

206 + 56

90 + 88*

O w i n g to the s p e c i e s d i f f e r e n c e s in the p a t t e r n of s t e r o i d b i o s y n thesis, M e t - e n k e p h a l i n , 10-5 mol/l, i n h i b i t e d the rate of a l d o s t e rone (Aldo), d e s o x y c o r t i c o s t e r o n e (DOC), c o r t i c o s t e r o n e (B), and c o r t i s o l (F) p r o d u c t i o n to d i f f e r e n t d e g r e e s in i s o l a t e d rat and b o v i n e a d r e n o c o r t i c a l cells. H o r m o n e c o n c e n t r a t i o n s (mean + S.D.) w e r e e x p r e s s e d as pg/8 x 105 c e l l s / 1 2 0 min. C a 2 + c o n c e n t r a t i o n in the m e d i u m w a s 7.62 mmol/l. S t a t i s t i c a l s i g n i f i c a n c e is i n d i c a t e d by asterisks (*p~0.O5; **p
(16, to

17)

prompted

enkephalins.

While Ca 2+

not in

us Table

affecting

the

to

medium,

II

study

the

shows

effect

the

results

Ca 2+ on of

these

response

experiments.

in

the

Met-enkephalin,

in

a concentration

of

2.54 of

mmol/l

1 0 -7

II

EFFECT OF MET-ENKEPHALIN

ADRENAL CELLS WITH DIFFERENT

presence

the

steroidogenesis

Table INHIBITORY

of

ON S T E R O I D B I O S Y N T H E S I S

Ca 2+ C O N C E N T R A T I O N S

2.54 m m o l / l

Ca 2+

OF RAT

IN THE M E D I U M

7.62 m m o l / l

Ca 2+

Aldo

85 + 18 %n.s.

63 + 12 %***

DOC

99 +

69 + 15 %**

B

95 + 16 %n.s.

4 %n.s.

88 +

5 %*

F i g u r e s r e p r e s e n t per cent (~ S.D.) v a l u e s of a d r e n a l a l d o s t e r o n e (Aldo), d e s o x y c o r t i c o s t e r o n e (DOC), and c o r t i c o s t e r o n e (B)production in r e s p o n s e to M e t - e n k e p h a l i n (10 -7 mol/l), w i t h 7.62 m m o l / l Ca 2+ in the m e d i u m , the i n h i b i t i o n s w e r e s i g n i f i c a n t s t a t i s t i c a l l y ( * p c 0.05; * * p < O.O1; * * * p ~ O.O01; n.s. = not s i g n i f i c a n t ) . (Baseline v a l u e s for all t h r e e s t e r o i d s w e r e e x p r e s s e d as iOO per cent (mean + S.D.).)

1350

Vol. 97, No. 4, 1980

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

F

B

+

+

L

300 •

200.

AIdo

%

%

% Io;~.-,[

,/,,.-t°';-t °s

/ ~*~000"

J

/

i

100

Is•

0 A C T H 1-24

50-

. . . .

I~ 1011 1010 109 108rn'~ f 1~1121~11 1~10 109 l(]Srnol ACTH 1-24 ACTHI-2'-

Fi 9 . 2.: In isolated bovine adrenal cells, with 2.54 mmol/l Ca 2+ in the medium, the inhibitory effect of Met- and Leu-enkephalin on steroid synthesis stimulated with ACTH was most pronounced for corticosterone (B) and cortisol (F), while the production of aldosterone (Aldo) was hardly affected. Solid line represents control (ACTH alone), dashed line, Met-enkephalin (10 -5 mol/l) + ACTH, dashed and dotted line, Leu-enkephalin (10-5 mol/l) + ACTH. Degree of significance: * p ~ 0 . 0 5 ; * * p ~ O . O O l ; n.s. = not significant. Baseline values (mean + S.D.) were as follows (in pg/8 x 105 cells/120 min) :--F, 182 ~ 27~ B, 1620 + 200; Aldo, 51 + ii.

mol/l,

significantly

B synthesis contained The steroid with

inhibitory synthesis

effect

was

administered

to 0 . 8 5

bathing

of

studied

Ca 2+ c o n c e n t r a t i o n

and to a l e s s e r

x 10 -8 m o l

the r a t e

of a d r e n a l

isolated

rat

Aldo,

adrenal

DOC

and

cells

Ca 2+.

of M e t - e n k e p h a l i n

a n d F, ously

the m e d i u m

7.62 m m o l / l

lower

mol/l

when

depressed

enkephalin during

in the m e d i u m

extent

apparent

stimulation

significantly

a l s o of A l d o ,

in d o s e s

(Fig.

in the b o v i n e

also when

with

ACTH.

Even

(2.54 m m o l / l ) , l O -5

reduced

ACTHI_24 2)

was

the

given

ranging

response

to s i m u l t a n e -

between

adrenal

of B

0.85

cell

x 10 -12

system.

DISCUSSION Endogenous physiological

opioid

peptides

regulation

are

thought

of p i t u i t a r y

1351

to be

hormone

involved

release

(18).

in the It has

VOI. 97, No. 4, 1980

also b e e n tides ses

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

shown that

traced

in the central

adrenotrophic

gested

to p l a y

further

the other

production

hand,

opioid

roid b i o s y n t h e s i s

agonists

have been

(22). W h e n e v a l u a t i n g biosynthesis borne

under

in m i n d

substances

according

(morphine,

The b i o l o g i c a l the adrenal

venous

la has not b e e n that these gical lation

the i s o l a t e d that tides

Met-

viously

on the adrenal enkephalins

could

the b l o o d

(21). On

stimulated conditions

ste-

with opioid

indicative

of a l t e r e d

steroid

however,

interfere

it should be (8), these

also w i t h

the adrenal

adrenal

cell

mechanism,

If the p e r i p h e r a l

regulation,

1352

theorize

via p h y s i o l o -

results

to the reguobtained

have

in

indicated

by the n e u r o p e p -

in a d d i t i o n through

to the pre-

a direct

adrenocortical

to be involved,

to the central

cortex

production

in

from the m e d u l -

one m i g h t

systems

is mediated,

hypophyseo-adrenal

detected

and c o n t r i b u t e

The p r e s e n t

hormone

release

to o r i g i n a t e

nevertheless,

adrenal

of steroid

could be shown

alternatively

cells

properties

stream,

biosynthesis.

cortex.

to s t i m u l a t e

inhibitory

of e n k e p h a l i n

reach

and L e u - e n k e p h a l i n s

known

(20),

found to inhibit

system

~9),

has corti-

culture

to other o b s e r v a t i o n s

yet,

rat and b o v i n e

the i n h i b i t i o n

sug-

same

and t h o u g h t

established

of c o r t i c o i d

the

pos-

release.

effluent

e.g.

cell

of opiates,

significance

substances

channels,

rat adrenal

beta-endorphine)

peptid

shown

experimental

to posses

experiments

cell

pep-

hormones

hypophysis

have b e e n

certain

the effects

that,

enkephalin-like

antagonists

shown

corticotrophic

has b e e n also

in i s o l a t e d

under

and CLIP h a v e b e e n

adrenal

in the rat adrenal

(22), moreover,

and the h y p o p h y s i s

from ovine

in fetal b o v i n e

beta-endorphine

corticosterone

adrenal

isolated

and m o r p h i n o m i m e t i c

system

AIpha-MSH

a role as fetal

properties

and s y n t h e t i c

nervous

properties.

beta-endorphine

cotrophic

ACTH

some of the A C T H - I i k e

effect

action

simultaneously

of

w i t h or

in the p h y s i o l o g i c a l

cont-

Vol. 97, No. 4, 1980

rol of adrenal

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

steroid synthesis,

a new aspect of neuroendocrine

regulation would possible emerge.

REFERENCES

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