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
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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.