Vol.86,

No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

January 15, 1979

B-ENDORPHIN STIMULATES

1-5

CORTICOSTERONE SYNTHESIS

IN ISOLATED RAT ADRENAL CELLS GOURI SHANKER and RAMESHWAR K. SHARMA Department of Biochemistry, Center for the Health Sciences, Received

October

20,

University Memphis,

of Tennessee Tennessee 38163

1978 SUMMARY

Studies costerone

synthesis

genesis data

are presented in isolated

has a lag

suggest

which

period

that

fasciculata

exhibits

hormone

that

cells.

of 3 to 5 minutes

B-endorphin

adrenocorticotropic

demonstrate

This

induces

activation

corti-

of steroido-

and is cycloheximide-sensitive.

steroidogenic

receptors

B-endorphin

activity

The

by binding

to the

of the cells.

INTRODUCTION Studies

by Mains

et.

al.

(1)

indicated

(ACTH) and B-endorphin

are derived

31,000

in the pituitary.

(2)

daltons

present

demonstrated

pituitary

that

gland.

mechanisms

endorphin

are

endorphin,

involved and

axis.

possibility.

We present

In

corticosterone

are it

secretion

identical.

This

influence

adrenal

the

present

study,

here

evidence

synthesis

adrenocorticotropic

common precursor

Subsequent

studies,

in the

ACTH, might

tary-adrenal

secrete

on these

common

like

a single

ACTH and B-endorphin

Based

regulatory

from

that

that

in response

studies

secreted has been

polypeptide

of Guillemin

postulated

the

isolated

rat

al

by the

(2)

that

the

of ACTH and 8-

possibility

steroidogenesis we have

of

et. -A

simultaneously

and biosynthesis raises

hormone

that

and thus

investigated fasciculata

B-

pitui-

the

first

cells

(3-5)

to B-endorphin.

MATERIALS AND METHODS The isolated method

of incubation

adrenal for

cells

were

prepared

ACTH, B-endorphin,

by trypsin or other

digestion

appropriate

(3-5). agents

The

was that

0006-291X/79/010001-05$01.00/0 1

Copyright All rights

@ 1979

by Academic Press, Inc. in any form reserved

of reproduction

BIOCHEMICAL

Vol. 86, No. 1, 1979

already

described

adrenals

from

mately

2 x lo6

buffer,

pH 7.4,

hours,

the

In general

(3,4).

16 rats

were

cells)

were

containing

from

was

States

Pharmacopeia.

All

isolated from

in

0.8

measured

chemicals

were

gland

After

(approxi-

incubating

(6). was used

was purchased

reagent

preparation,

ml of Krebs-Ringer-bicarbonate

3465.4)

Standard,

cell

each adrenal

fluorometrically

weight

Pharmacopeia

adrenal

and 0.2% glucose.

(Molecular

other

each

resuspended 4% albumin

Calbiochem

ACTH, a United

for

used and the cells

corticosterone

endorphin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

grade

for

Synthetic

in these from

and were

2 8-

studies.

United

States

obtained

commer-

cially.

RESULTS AND DISCUSSION Fig. typical

I

shows

sigmoid

with

the

concentration

(30x10-lLM)

of

that

with

steroidogenic increase

study

measures

potency

of

affinity

the

binding

lOOO-fold

lower

than

higher

significant

than

that

maximal

decline

reduction

in corticosterone

the

obtained

of

value the

membrane

with receptor

causes

or --in vivo the

of

receptor

B-endorphin

with

An interesting

steroidogenic

cell

cell

observation

B-endorphin

There

by 1 X 10e6Mof 8-endorphin

x 10m7M.

This

by B-endorphin.

The level

which

these is

study

was

caused

was almost

modification such

a

a 50%

as compared

an allosteric at

on

receptors

of this

synthesis

indicates

preparation

Based

adrenal

(3-5)

Therefore,

cell

level.

synthesis.

(6-fold)

(7).

this

of

the maximal

preparation

activity

concentrations

corticosterone

steroidogenesis

significant

adrenal

ACTH

as ACTH in

Nonetheless,

experiments,

cell

than

as effective

a very

of

steroido-

higher

the maximal

any ACTH-degrading

of ACTH.

2.2

0.1%

The isolated

ACTH at

of peak

lOOO-fold

of ACTH.

B-endorphin

slices

is

in a

affinity

The half-maximal

only

one-sixth

formation.

adrenal

criteria,

is

production

the binding

Furthermore,

does not possess

to the the

(40x10-'E(I)

only of

of corticosterone

in contrast

that

is

indicating

receptors.

B-endorphin

concentration

in this

cell

production.

B-endorphin

corticosterone

manner

B-endorphin

corticosterone

obtained

stimulates

fasciculata

indicating

activating

used

B-endorphin

concentration-response

the enkephalin genie

that

a negative

to

Vol. 86, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig.

in isolated

fasciculata

1:

Steroidogenesis

cells

in response

to B-endorphin

and ACTH. Incubation

System:

dissolved

4% albumin

Incubation

values trol

values

was for

known

takes

about

the

2 hours.

nature

cannot

of the

Total

Results

determinations

curves

place

suspension,

0.8 ml;

or Krebs-Ringer-bicarbonate

from

have been subtracted

tration-response

cell

and 0.2% glucose.

of 6 separate

cooperativity

adrenal

in 0.2 ml vehicle

containing ml.

isolated

from

buffer, volume

are

as the

experiments.

all

experimental

have been plotted

on log-log

interaction

pH 7.4,

of incubation,

expressed

3 different

be identified

reagents

results.

1 mean Con-

Concen-

scale.

at this

time,

since

of 8-endorphin

with

nothing

fasciculata

is cell

receptors. To answer genesis

through

determined

by

concentrations ing

that

combining

the

question

B-endorphin

the common or different incubating of these

8-endorphin with

of whether

the agents. does

not

the ACTH receptors

cells

receptors, with

The results have

its

(Table

corticosterone combined

obtained

steroido-

production

maximal

The alternative

was

steroidogenic

were not additive,

own distinct 1).

3

the

and ACTH activate

receptors possibility

suggestbut

acts

by

is that

BIOCHEMICAL

Vol. 86, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE Effect

of

adrenal

Cycloheximide

cells

in

on the

response

to

1

stimulation

of

B-endorphin

and

steroidogenesis

in

ACTH.

ADDITIONS

CORTICOSTERONE (ug/2 h-1

0.045

Control B-endorphin

0.290

(Z.ZX~O-~M)

ACTH (100

vu/ml)

1.700

Cycloheximide

(10 uM)

ACTH (100

uU/ml)

B-endorphin

0.001

+ B-endorphin

(2.2

ACTH (100

x 10-/M)

(2.2

Cycloheximide

ACTH (100 VU/ml) + Cycloheximide

+ B-endorphin (10 uM)

Incubation

system:

dissolved

in

containing

ml

values

experiments.

uM)

0.019 0.260

x 10V7M)

adrenal or

are

Control

(10

0.270 -

and 0.2%

shown

1.660

uM)

(2.2

vehicle

4% albumin

mean

(10

Isolated

0.2

x 10s7M)

+ Cycloheximide

uU/ml)+

The

isolated

cell

suspension,

0.8

Krebs-Ringer-bicarbonate glucose.

derived

value

-

Total from

has

buffer,

volume

of

subtracted

reagents pH

incubation,

6 observations

been

ml;

from

7.4, 1 ml.

from

3 different

all

experimental

results.

B-endorphin these

and

data,

ACTH

one

could

endorphin

is

the

analogous

to

the

definite

lag

cyclic in

in

and

ACTH-induced

data,

one the

same ACTH of

(Table

GMP

could

propagation

separate

postulate

period

mide-sensitive

have

cyclic

as

receptors

that that

effect

of

of

This

5 minutes

(Fig.

2).

1).

To

the

majority

date, of (for

that B-endorphin

cyclase

indeed

may

B-endorphin-stimulated

3 to

steroidogenesis

common

steroidogenic-activating

ACTH.

(3),

AMP components

postulate

the

but

these

the

a review cyclic action.

4

of play

see

process

of

the

since

ref.

nucleotides

also

evidence

an important 8). also

Based have

From

mechanism case

steroidogenesis This

cell

be

system.

has is

cyclohexi-

indicates

that

mediatory

role

on

the

a similar

present role

8-

a

BIOCHEMICAL

Vol. 86, No. 1, 1979

I

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

II

rig

I

I

I

I

I-

0.30 &I OaT8 t

0.20

-

% e b 0.15

-

% Q 0.10

-

0.05

-

0

/ 0

/*

I IS

I

I

30

I

I

60

I

so

120

I 150

TIME(MINUTE6)

Fig.

2:

Time

course

response Fig.

study

of

steroidogenesis

to B-endorphin.

Conditions

in

isolated

fasciculata

of the experiment

were

cells

in

identical

to

1.

ACKNOWLEDGMENTS This and National

work was supported Science

by grants

Foundation

from National

Cancer

Institute

(CA-16091)

(PCM 7800860). REFERENCES

1.

Mains, U.S.A.

R. E., Eipper, 2, 3014-3018.

8. A. and Nicholas,

2.

Guillemin, R., Vargo, T., Rossier, J., Minick, S., Vale, W. and Bloom, F. (1977), Science 197, 1367-1368.

3.

Kitabchi,

4.

Sharma, R. K., 994-1003.

5.

Sayers, G., Swallow, g, 1063-1068.

6.

Glick,

7.

McMartin, Brugger,

8.

Sharma, R. K. (1978), K. and Criss, W. E.),

A. E. and Sharma,

D.,

Hashimoto,

Von Redlich,

L. (1977),

R. K. (1971),

L.

and

N.

S. (1964),

C., Purdon, G. E. E., Schenkel, L., P. (1977), M., Rittel, W., and Sieber, "Endocrine Control In: pp. 13-52, Raven Press,

c

Ling,

N.,

g,

A. E. (1972),

Giordano,

D. and Levine,

Natl.

Endocrinology

K. E. and Kitabchi, R.

Proc.

D.

Acad. Rivier,

Sci. C.,

1109-1116, Endocrinology%,

(1971),

Endocrinology

Endocrinology

74,

653-655.

Desaules, P. A., Maier, R., J. Endocrinol. 73, 79-89. in Neoplasia" New York.

(eds.

Sharma,

R.