COMPARED EFFECTS OF ACTH, ANGIOTENSIN II AND POMC PEPTIDES ON ISOLATED HUMAN ADRENAL CELLS
M. T. PHAM-HUU-TRUNG, A. BOGYO, P. LENEUVE and F. GIRARD INSERM U 142, Hôpital Trousseau, Paris, France
Summary-Human adrenocortical tissue obtained, on eight occasions, at the time of nephrectomy for renal carcinoma (outside the adrenal pole) was treated by collagenase to dissociate the cells. These were then submitted to a short, 2-h, incubation with the N-terminal fragment (16 K) of POMC, its derivative, “3-MSH, B-lipotropin and ß-endorphin, in parallel with ACTH 1-24 (Synacthen Ciba) and angiotensin II (AII, Hypertensin Ciba).
Under the influence of ACTH (10-10 M), and AII (10-10 M), basal glucocorticoid output, including more than 80% cortisol, was increased by factors of 3 + 0.51 (SEM) and 1.35 ± 0.12 (SEM), respectively. The corresponding aldosterone responses were 1.60 ± 0.13 for ACTH and 1.38 ± 0.09 for AII. With the exception of y3-MSH, the POMC peptides under study had no steroidogenic effect. 73-MSH (10-9 M) and AII (10-10 M) stimulated aldosterone production to approximately similar levels of. respectively, 1.23 ±0.05 and 1.38 ±0.09 times the basal production. In contrast to All however, y3-MSH showed no apparent effect on glucocorticoid output. Steroidogenic response to ACTH was potentiated by y 3-MSH at a concentration of 10-10 M which, when used alone, proved ineffective. This potentiating effect was pronounced for the aldosterone response, whereas the glucocorticoid production was hardly affected. This action ceased to be visible when the cells reached maximal stimulation by ACTH.
These findings suggest that y3-MSH-a portion of the 16 K fragment-may have a possible role in aldosterone secretion.
INTRODUCTION
It is now well established that ACTH derives from a precursor- proopiomelanocortin-or POMC, con- taining lipoprotein/endorphin as the C-terminal por- tion, and a glycopeptide of 109 aminoacids as the N-terminal portion. The latter comprises the 16 K fragment-76 aminoacid long-and a joining peptide of 30 aminoacids occurring between 16 K and ACTH [1, 2]. Furthermore, the 16 K fragment con- tains a melanotropin sequence denoted as y3 MSH.
The effects of B-lipotropin, (B-LPH), B-endorphin, (ß-end), 16 K and its y3-MSH derivative on animal adrenal steroidogenesis have been studied [3-8]. The reactivity of human aldosteronomas to 16 K and 73-MSH has been described [9-11]. Nonetheless, there are very few reports of studies wherein normal human adrenals have been utilized [6]. The use of isolated cells from adrenals, removed on the occasion of nephrectomy for renal carcinoma, should prove enlightening as to the action of these peptides on man’s adrenal steroidogenesis.
EXPERIMENTAL
On eight occasions, adrenals were obtained as nephrectomies for renal carcinomas (outside the ad- renal pole) had just been performed. They were treated in accordance with a protocol described elsewhere [12, 13]. Briefly, the adrenal fragments were submitted to two 15-min digestions in Eagle’s min- imum essential medium, containing 2 mg/ml bovine
serum albumin (MEM-BSA) and 2 mg/ml col- lagenase (Sigma) [2 mg/g tissue]. After each digestion, the supernatant was discarded and the cells were dissociated by drawing up and expelling the adrenal pieces through a polyethylene tube (2 mm dia) at- tached to a 10 ml syringe, into MEM-BSA contain- ing a 1 mg/ml Lima bean trypsin inhibitor. The resulting cells were filtered, centrifuged at 200 g for 20 min, washed with MEM-BSA and centrifuged again. They were subsequently re-suspended in MEM-BSA and distributed into Teflon beakers con- taining a 0.9 ml cell suspension (4-5 × 105 cells) each. A 1-h pre-incubation at 37 ℃ under an atmosphere of 95% O2 and 5% CO2 preceded the process of adding the hormones to be tested. ACTH (Synacthen, Ciba), angiotensin II (AII) (Hypertensin, Ciba) and the POMC peptides under study, were dissolved in a saline solution containing 0.9% NaCl and 2 mg/ml bovine serum albumin adjusted to pH 3.2 with 1 N HCI. The concentrations being assayed were added in 0.1 ml vol. All experiments were carried out in at least duplicate and the samples were incubated for 2 h. The purified human 16 K fragment, synthetic 73-MSH and human purified LPH were generously donated by Drs P. J. Lowry (St Bartholomew’s Hospital, London), N. Ling (Salk Institute, La Jolla, Calif.) and D. N. Orth (Nashville, Tenn.), re- spectively. Human synthetic Bendorphin was pro- vided by Beckman.
One ml incubation volume containing the steroids produced under the influence of ACTH, AII and POMC peptides was extracted by 10 ml dichlo-
romethan (Merck, Darmstadt). Glucocorticoids were measured by a competitive protein binding technique [14] and aldosterone by a direct radioimmunoassay [15].
RESULTS
The spontaneous glucocorticoid output varied widely from one experiment to another: 153 ng ± 6 (SEM, n = 4) to 2,015 ng/ml ± 63 (SEM, n = 4) with a geometric mean of 669 ng/ml, (Fig. 1). In two experiments, the ratios of corticosterone to cortisol were determined in basal conditions and were found as: 0.114 and 0.167. The spontaneous aldosterone output ranged from 0.215 ng/ml ± 0.05 (n =2) to 3.37 ± 0.074 (n =4) with a geometric mean of 0.926 ng/ml. Consequently, the steroidogenic re- sponses to the hormones under study were expressed in terms of the ratio of the stimulated level to the basal level, whenever several experiments were con- sidered.
16 K, 73-MSH, B-LPH and B-end were initially examined for their steroidogenic properties at single doses of 2.5 ng or 5 ng [Fig. 2, Table 1], (approx 10-10 M-10-9 M). Of these, only y 3-MSH exhibited a significantly visible effect in aldosterone production though not in glucocorticoid production. No stimu- lation could be seen, even when the 16 K and B-LPH concentration was doubled. Our study was therefore focused upon the effects of y3-MSH on non- tumorous human adrenal cells.
As shown in Fig. 3, 10-10 M ACTH and 10-10 M AII increased the basal glucocorticoid output by factors of 3±0.51 (SEM) and 1.35±0.12, re- spectively. The corresponding mean aldosterone re- sponses were 1.60 ±0.13 for ACTH and 1.38 ± 0.09 for AII. 10-9 M y3-MSH stimulated aldosterone production to an extent close to that observed with
ng / ml
ng/ml
1000
1.0
.
100
0.1
Glucocorticoid
Aldosterone
Basal output
10-10 M AII: 1.23 ±0.05, while no apparent effect was noted upon glucocorticoid production.
Beyond the 10-9 M concentration, y3-MSH ex- hibited an evident steroidogenic potency which was visible in both glucocorticoid and aldosterone prod- uction (Fig. 4).
In two out of three experiments, 10-10 M poten- tiated the action of ACTH on aldosterone prod- uction, though not on glucocorticoid production (Fig. 5). In the third experiment, wherein aldosterone output was already maximally stimulated, a higher 23-MSH concentration (10-9 M) was ineffective.
Glucocorticoids (ng /ml )
Aldosterone (ng/ml)
ACTH
. AI
· 16K
· Y3 MSH
· BLPH
3000
3
Production
… .
2000
2
+2SD
La
-2SD
1000
1
+ 2SD
4
-2SD
2.5
250
2500
2.5
250
2500
pg
| ng/ml | |||||
|---|---|---|---|---|---|
| Peptide | 0 | 0.025 ACTH | 2.5 AII | 5 BLPH | 5 BEND |
| Gluco. | 153 ± 8* | 633 ± 41 | 295 ± 25 | 178 ± 19 | 170 ±23 |
| Aldo. | 3.05 ± 0.89 | 5.06 ±0.41 | 5.15±0.50 | 2.68 ± 0.63 | 3.13 ± 1.16 |
| n = 4 | n =7 | n = 2 | n = 4 | n = 3 | |
*SEM.
DISCUSSION
The results, that we have been able to obtain from an important collection of human adrenals, highlight a certain number of points. As expected, the relative response of glucocorticoids to ACTH is greater than that of aldosterone. More remarkable still is that the glucocorticoid and aldosterone responses to AII should be of the same order of magnitude. It is thus quite clear that AII also stimulates glucocorticoid production (corticosterone + cortisol) albeit its effect is modest when compared with that of ACTH. As often occurs with in vitro systems, AII is less effective
Glucocorticoids
Aldosterone
Stimulated level / Basal level
· ACTH
. AH
3
Y3 MSH
2
1
12
11
10
9
12
11
10
9
-Log 10 [peptide ] M
PRODUCTION
GLUCOCORTICOIDS (ng/ml)
ALDOSTERONE (ng/ml)
ACTH
ACTH
1000
1
8 3 MSH
X 3 MSH
0
0
10
9
8
7
10
9
8
7
-LOG 10 [ PEPTIDE ] M
than ACTH in the stimulation of aldosterone prod- uction, thus raising the question of a necessary cooperation between these two hormones to maintain aldosterone secretion [16, 17].
Our option to limit the concentrations of the POMC derived peptides to 10-10-10-9 M levels, when testing them in parallel with ACTH and AII, has been deliberate. This is perhaps the reason why we have not observed any steroidogenic effects with 8-LPH. 16 K and ß-end, in contrast with some studies which used higher concentrations when experimenting with animals or men [3-7]-despite the fact that these concentrations are hardly compatible with plasma concentrations encountered in the human blood: of the order of 10-12 M-10-10 M [18-20]. This being said, it is certainly with y3-MSH that the most consistent results were obtained. At a 10 -9 M concen- tration, y3-MSH stimulates aldosterone production and raises it to a level close to that obtained with 10-10 M AII. Yet, there is no apparent effect upon glucocorticoid production. Beyond a 10-9 M concen- tration, y 3-MSH reveals obvious steroidogenic prop- erties which have not been observed with animal adrenal cells [5, 21]. With such high concentrations the relative effects of y3-MSH on glucocorticoid and on aldosterone production are comparable, as is also the case with AII when used at lower concentrations.
GLUCOCORTICOIDS (ng/ml)
ALDOSTERONE (ng/ml)
5,000
5
+ 83 MSH
10-9M
2,000
2
+ 63 MSH
10-10M
1,000
1
+6 3 MSH
10-10M
PRODUCTION
0.5
12
11
10
9
12
11
10
9
-LOG 10 [ACTH] M
The potentiating effect of y3-MSH is pronounced for the aldosterone response to ACTH, whereas the glucocorticoid production is hardly affected. This action ceases to be visible when the cells are already maximally stimulated. This series of experiments are in conformity with the observations by Farese et al.[21].
Results, as we have obtained from non-tumorous human adrenal cells, confirm those of an earlier work devoted to human aldosteronoma cells [11]. y3-MSH, when used alone or in association with ACTH will, in preference, stimulate aldosterone secretion. At high concentrations it has steroidogenic properties of its own. However, the mechanism whereby it potentiates ACTH-induced aldosterone output and provokes steroidogenesis is a matter for further investigation.
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