Dazord, A.

Gallet, D.

Saez, J.M.

Elsevier - Publisher Connector 

Protein degradation in adrenal cells in culture is inhibited by ACTH and cyclic AMP 

Volume 83, number 2 FEBS LETTERS November 1977 
PROTEIN DEGRADATION IN ADRENAL CELLS IN CULTURE IS INHIBITED BY ACTH 
AND CYCLIC AMP 
A. DAZORD, D. GALLET and J. M. SAEZ 
Unit4 de Recherches sur le Contr6le Hormonal des Activitks Cellulaires, INSERM, U. 162, Hdpital Debrousse, 29 Rue Soeur 
Bouvier, 69322 Lyon Cedex 1, France 
Received 2 September 1977 
1. Introduction 
In vivo trophic effects of adrenocorticotropic 
hormone (ACTH) are well known [l] . Most authors 
have found that ACTH treatment in vitro stimulates 
total protein synthesis [2-41 . But the mechanism of 
this action is unknown. We recently showed that in a 
functional cloned mouse adrenal cell line (Yl) ACTH 
and all compounds able to increase cyclic adenosine 
3’5’-monophosphate (CAMP) induce a dramatic 
increase of the incorporation of radioactive leucine 
into a small peptide (mol. wt 3500) [5]. But this 
increase cannot explain the enhanced rate of total 
protein synthesis produced by ACTH. Likewise the 
stimulatory effect of ACTH on other specific adrenal 
proteins [6,7] is not sufficient to account for its 
effect on total adrenal protein synthesis. 
One could therefore postulate that ACTH action 
on protein synthesis involves alterations in the rate 
of degradation of adrenal proteins. The present paper 
reports for the first time that corticotropin l-24 
tetracosapeptide (ACTH1_-24), two of its analogues: 
corticotropin-(11-24)-tetradecapeptide (ACTH11_24) 
and (9-tryptophan(o-nitrophenyl sulfenyl) )-cortico- 
tropin (l-24)-tetracosapeptide (NPS-ACTHI -24) 
and dibutyryl cyclic adenosine 3’5’:monophosphate 
(dbcAMP) are able to inhibit in vitro adrenocortical 
protein degradation. 
2. Materials and methods 
ACTHI _-24 was provided by Ciba. NPS-ACTHI -24 
and ACTHll_2; were a gift from Drs Rittel and 
North-Holland Publishing Company - Amsterdam 
Desaulles (Ciba-Geigy AG, Basel, Switserland). [ 3H] - 
Leucine (spec. act. 25-35 Ci/mmol) was obtained 
from Saclay, France. 
Functional cloned mouse adrenal tumor cells (Y1) 
developed in the laboratory of G. Sato [8] were 
purchased from the American Culture Cell Repository 
(Rockville, MD). They were grown in Ham’s F-10 
medium supplemented with 10% heat-inactivated 
horse serum and 2.5% heat-inactivated fetal calf serum 
(FCS). 
Protein degradation was measured in the cells 2 
days after seeding the same amount of cells (5 X lo6 
cells) in 25 cm2 Falcon flasks. They were first 
incubated for 15 h with 15 /.&i/ml [3H]leucine, 
0.1 mM, in Ham’s F-10 supplemented with 0.2% 
FCS. At the end of the labeling period the cells we;e 
rinsed twice with 3 ml Ham’s F-10 supplemented with 
0.2% FCS and 5 mM leucine, and incubated 3 h in 
this medium. At the end of this chase period they 
were incubated in 3.3 ml of the same medium in the 
presence or absence of hormone or dbcAMP. In these 
conditions of serum deprivation it has been shown 
that these cells are synchronized in G1 [9]. 
Repeated samples of medium (100 4) were taken 
from the same flask during the experiment, precip- 
itated by equal vol. 20% trichloroacetic acid (TCA) 
in the presence of 10 ~1 10 mg/ml bovine serum 
albumin. After centrifugation, 180 fi supernatant 
were counted in 5 ml scintillation fluid (8.25 g Scinti- 
mix, 500 ml Triton X-100 in 1 litre toluene). Each 
experimental point was run in triplicate, and each 
experiment was performed at least 3 times. Measure- 
ment of the trichloroacetic acid-soluble radioactivity 
in the medium of cultured cells has been shown to 
307 
Volume 83, number 2 FEBS LETTERS November 1977 
give a good estimation of protein degradation in 
cultured cells, especially in turnover experiments 
[lo] . Nevertheless at the end of the sampling period, 
the cells were rinsed and solubilized as described 
[5,6] and the TCA-soluble radioactivity in the cell 
was measured. 20a-hydroxypregn4-ene-3-one 
(20aOH progesterone) was measured by a radio- 
immunoassay (antibodies of E. G. Abraham: S1556#5) 
following a method described [ 1 l] except that the 
steroid was measured directly in the medium without 
prior fractionation in a celite column. 
3. Results and discussion 
After a labeling period of 15 h and a chase period 
of 3 h the adrenal cells were incubated with two dif- 
ferent concentrations of ACTH, lo-* M and 10e6 M, 
in the presence of 5 mM leucine during 27 h (flg.1). 
After 40 min incubation, the TCA-soluble radio- 
activity of the ACTH-treated cells represents 98% of 
IO0 200 
I 
1 3 6 121 27 
TIME ~HOURS) 
Fig.1. Effect of ACTH on the release of TCA-soluble radio- 
activity in the medium after the chase period (mean of 
triplicates r SEM). (o-0) Control; (m..g) ACTH, _-2, 10e6 M; 
(b-u) ACTH,_,, lo-* M; (A) hCG 10-s M. The bars represent 
the 20olOH progesterone ng/ml secreted in the medium at the 
end of the 27 h incubation (mean * SEM of triplicate flasks). 
the controls. But after 6 h incubation, the inhibition 
of the degradation is obvious since the TCA-soluble 
radioactivity in the medium of the ACTH-treated cells 
represents 80% control. After 20 h incubation, the 
inhibition induced by the hormone reaches a plateau. 
The TCA-soluble radioactivity in the medium of the 
treated cells represents 75% control. Other poly- 
peptide hormones, like human chorionic gonado- 
trophin (hCG CR 115 provided by NIH, at a concen- 
tration of lo-’ M), have no effects. 
The same experiment was undertaken in the 
presence of dbcAMP 1 mM, and 20aOH progesterone 
100 ng/ml which is the main steroid produced by this 
cell line. Figure 2 shows that dbcAMP but not 
20crOH progesterone has an inhibitory effect on protein 
degradation. In the same experiment the TCA-soluble 
radioactivity of the solubilized cells was measured at 
the end of the 24 h incubation. As shown in table 1, 
the TCA-soluble radioactivity of the ACTH- and 
dbcAMP-treated cells is significantly lower than in 
the control conditions. 
!OOO 
1 5 9 ’ 
TIME (HOURS1 
Fig.2. Effect of ACTH, dbcAMP, 20oOH progesterone on the 
release of TCA-soluble radioactivity in the medium after the 
chase period (mean of triplicates f SEM). (o-0) Control; 
(m..=) ACTH,_,, 10m6 M; (o-n) dbcAMP 1 mM; (a) 20aOH 
progesterone 100 ng/ml. The bars represent the ZOoOH 
progesterone secreted in the medium at the end of 24 h 
incubation (mean + SEM of triplicate flasks). 
308 
Volume 83, number 2 FEBS LETTERS November 1977 
Table 1 
TCA-soluble radioactivity in solubihzed cells at the end of 
the sampling period described in fig.2 (mean * SEM of 
triplicate flasks) 
cpm/pg proteins 
Controls 1850 f 20 
ACTH 1O-6 M 1620 f 30 
dbcAMP 1 mM 1530 t 31 
20olOH Progesterone 1860 f 25 
100 ng/ml 
In these two experiments the labeling time was 
15 h. The proteins whose degradation was inhibited 
by both ACTH and dbcAMP have a long turnover rate. 
Figure 3 shows that both ACTH and dbcAMP also 
inhibit the degradation of proteins with rapid rates of 
turnover. In this experiment the cells were incubated 
for 2 h with [3H]leucine 20 @/ml, rinsed twice with 
the chase medium, and immediately incubated with 
TIME (HOURS1 
Fig.3. Effect of ACTH and dbcAMP on the release of TCA- 
soluble proteins after a two hours labeling period. Mean of 
triplicates + SEM. (o-0) Control; (=..m) ACTH, _-24 10e6 M; 
(o-o) dbcAMP 1 mM. The bars represent the 20aOH 
progesterone secreted in the medium at the end of the 
28 h incubation Cmean 2 SEM of triplicate flasks). 
ACTH and dbcAMP in the presence of 5 mM leucine 
(no chase period). 
The present results (fig.1 -fig.3) show that ACTH 
and dbcAMP decrease the degradation of adrenal 
proteins with both long and short turnover rates. The 
quantitative significant difference observed between 
the action of ACTH and dbcAMP after 9 h incubation 
could be due in part to the fact that most of the 
hormone is degraded by the cells after 8 h incubation 
(unpublished data). Nevertheless our results suggest 
that CAMP formation could be an obligatory step for 
ACTH-inhibited adrenal proteolysis. To check this 
hypothesis the effects of two analogues of ACTH 
were investigated: 
ACTHI l-24 
NPS-ACTHr --24 
which is a pure antagonist of 
ACTHi _-24 concerning CAMP and 
steroid production, and which is able 
to bind to adrenal plasma membranes 
[12-141. 
which binds to adrenal plasma mem- 
branes with the same affinity as 
ACTH1_24 but which has a lower 
biological effect [ 12-141 . 
Figure 4 shows that cyclic AMP is probably not the 
only factor involved in the inhibition of adrenal protein 
degradation for two reasons: 
1. ACTH11--24 though less effective than ACTH,_,4 
and NPS-ACTHr _-24 has a significant inhibitory 
effect. 
2. On the other hand NPS-ACTH1_24 was used at a 
higher concentration than ACTHi _24. Nevertheless 
at this concentration the effect on steroidogenesis 
was less important than with ACTHi _-24, and the 
production of cyclic AMP is known to be less than 
1% of maximum cyclic AMP production [ 141. 
Despite a smaller biological effect on CAMP and 
steroid production, this analogue has a higher effect 
on protein degradation. 
These results show that the C-terminal sequence of 
ACTH in addition to being responsible for the high 
309 
Volume 83, number 2 FEBS LETTERS November 1977 
!OOO 
CONTROL 
CONTROI 
m 
1 3 5 //2l 23 21 
TIME C HOURS, 
‘1.24 
2oa 
Fig.4. Effect of ACTH and its analogues on the release of 
TCA-soluble radioactivity. Cells were labelled for 15 h, rinsed 
and incubated in the chase medium as described in section 2, 
then incubated with the hormones. (e-0) Control; (m..m) 
ACTH,, w-2, 2 . 1O-5 M; (o--o) ACTH,_,, 1O-6 M; (e-e) 
NPS-ACTH 1 _-2, 3 . lo-’ M. The bars represent the 20oOH 
progesterone secreted in the medium at the end of 27 h 
incubation (mean + SEM of triplicate flasks). 
affinity of the hormone for its receptor, has some 
biological effects similar to those of ACTH1_24. 
This paper represents the first evidence that ACTH 
can directly inhibit adrenal protein degradation. Our 
results are in agreement with the work of Cannick and 
Villee [ 151 who showed indirectly that when ACTH 
has been chronically administered to rats the mean 
half-life of total adrenal proteins is significantly longer 
in ACTH-treated animals. This inhibition cannot com- 
pletely account for the stimulation of protein synthesis 
we observed in these cell lines [4,5] for several 
reasons: 
1. Stimulation of total protein synthesis of these cells 
is unaffected by cyclic AMP and by ACTH11_24 or 
NPS-ACTHr _24 [4] . 
2. Stimulation of protein synthesis by ACTH is maxi- 
mal after 7 h incubation in the presence of ACTH, 
then decreases [4]. On the contrary our results 
show that inhibition of the proteolysis by ACTH 
reaches a plateau after 20 h incubation. 
Studies are in progress in order to elucidate the 
mechanisms of this inhibitory effect and determine 
which proteins are protected from degradation by 
ACTH. 
Acknowledgements 
We thank Miss Joelle Bois for secretarial assistance. 
This work was supported by grants from Institut 
National de la Sante et de la Recherche Medicale 
(ATP No. 49.77.81) Delegation G&r&ale a la Recher- 
the Scientifique et Technique (76.7.0802) and la 
Ligue Nationale Contre le Cancer. 
References 
[l] Imrie, R. C., Ramaiah, T. R., Antoni, F. and Hutchinson. 
W. C. (1965) J. Endocrinol. 32,303-312. 
[2] Koritz, S. B., Peron, F. G. and Dorfman, R. I. (1957) 
J. Biol. Chem. 226,643-650. 
[3] De Nicola, A. F., Clayman, M. and Johnstone, R. M. 
(1968) Endocrinology 82,436-446. 
[4] Dazord, A., Gallet, D. and Saez, J. M. (1977) CR Acad. 
Sci. Paris 284, 1309-1312. 
[5] Dazord, A., Gallet, D. and Saez, J. M. (1977) Biochem. 
Biophys. Res. Commun. 76, 1238-1246. 
[6] Kowal, J., Simpson, E. R. and Estabrook, R. W. (1970) 
J. Biol. Chem. 245,2438-2443. 
[7] Grower, M. F. and Bransome, E. D. (1970) Science 168, 
483-485. 
[8] Buonassissi, V., Sato, G. and Cohen, A. I. (1962) Proc. 
Natl. Acad. Sci. USA 48, 1184-1190. 
[9] Weidman, E. R. and Gill, G. N. (1977) J. Cell. Physiol. 
90,91-103. 
[lo] Poole, B. and Wibo, M. (1973) J. Biol. Chem. 248, 
6221-6226. 
[ 1 l] Saez, J. M., Morera, A. M., Dazord, A. and Bertrand, J. 
(1972) J. Endocrinol. 55,41-49. 
[ 121 Saez, J. M., Morera, A. M., Dazord, A. and Bataille, P. 
(1974) J. Steroid Biochem. 5,925-933. 
[ 131 Saez, J. M., Dazord, A., Morera, A. M. and Bataille, P. 
iI41 
1151 
(1975) J. Biol. Chem. 250, 1683-1689. 
Seelig, S. and Sayers, G. (1973) Arch. Biochem. Biophys. 
154,230-239. 
Canick, J. A. and Villee, D. B. (1974) Biochem. J. 144, 
397-403. 
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Protein degradation in adrenal cells in culture is inhibited by ACTH and cyclic AMP

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