Effects of pro-opiomelanocortin-derived peptides on adrenal steroidogenesis in guinea-pig adrenal cells in vitro

J. Steroid Biochem. Molec. Biol. Vol. 44, No. 1, pp. 77-83, 1993 Printed in Great Britain

EFFECTS PEPTIDES

OF

0960-0760/93 $6.00 + 0.00 Pergamon Press Ltd

PRO-OPIOMELANOCORTIN-DERIVED

ON

GUINEA-PIG

ADRENAL

STEROIDOGENESIS

ADRENAL

CELLS

IN

IN

VITRO

YVONNEO'CONNELL,T. JOSEPHMCKENNA and SEANK. CUNNINGHAM* Department of Endocrinology,St Vincent's Hospital, Dublin 4, Ireland

(Received 23 March 1992;accepted 25 September 1992) Summary--The specific control of adrenal androgen secretion is unclear. This study was undertaken to investigate the effects of peptides derived from the ACTH precursor molecule pro-opiomelanocortin (POMC) on cortisol and androgen production using isolated guinea-pig adrenal cells. ACTH 10-13-10-9M, stimulated steroid production in a dose dependent manner, reaching a maximum of 12, 10 and 7 times basal levels for cortisol, androstenedione (A4) and dehydroepiandrosterone (DHEA), respectively, measured by specific radioimmunoassays, fl-Lipotropin (fl-LPH), 10-~°-10-s M, also stimulated steroid production, reaching 6, 5 and 5 times basal levels of cortisol, A4 and DHEA, respectively. The N-terminal 16K fragment, Y3-and fl-MSH stimulated steroid production which reached statistical significance (P < 0.05) only in the case of cortisol. Joining peptide, ~-, fl- and 7-endorphin resulted in no significant change in steroid production. Met- and leu-enkephalin resulted in significant inhibition of DHEA production. POMC-derived peptides altered the steroid response to ACTH. fl-LPH and 16K fragment significantly increased the eortisol and A4 responses to a low concentration of ACTH. fl-LPH reduced the maximum cortisol and A4 responses to a high concentration of ACTH. This study suggests that fl-LPH may have a role in modulation of adrenal steroidogenesis but yielded no evidence to support a role for POMC-derived peptides in preferential stimulation of adrenal androgen production in guinea-pig adrenal cells.

INTRODUCTION

adrenal cortex with its functional, biochemical and morphological similarities to the human gland [8, 9] was considered to be a suitable model for investigation of the factors controlling adrenal androgen production. In this study we have used a guinea-pig adrenal cell preparation to examine the effects of POMCderived peptides on cortisol and androgen production.

The control of adrenal androgen production has not been elucidated[l]. Adrenocorticotropin (ACTH), which has a well established role in regulating glucocorticoid production [2], is also involved in the regulation of adrenal androgen production [3]. However, there is evidence that in primates some factor in addition to A C T H is also involved in the regulation of adrenal androgen production [1, 3, 4]. It has been suggested that the proposed adrenal androgen stimulating factor may be related to the A C T H precursor molecule, proopiomelanocortin (POMC) [1, 5, 6]. POMC, a 265 amino-acid polypeptide, as well as giving rise to ACTH, fl-lipotropin (fl-LPH) and flendorphin (fl-END), is also the precursor of a 16kDa N-terminal fragment (16K fragment), other endorphins, a family of melanocyte stimulating hormones (MSHs), enkephalins (ENKs) and joining peptide (JP)[7]. In the absence of suitable human adrenal tissue, the guinea-pig

METHODS

Optimization of cell suspension preparation Adrenal cell suspensions were prepared using guinea-pig adrenals. The chopped adrenal tissue was subjected to collagenase treatment and physical dispersion. Isolated adrenal cells were harvested after washing and centrifugation before final resuspension in medium. Pre-treatment with ACTH, i.e. a single injection s.c. of long-acting A C T H (Acthar Gel, Rorer, England, 0.2ml), 3 days prior to adrenalectomy, was found to increase responsiveness to A C T H stimulation for both cortisol and androstenedione (A4) production,

*To whom correspondenceshould be addressed. 77

YVONNE O'CONNELL et al.

78

while basal production remained unchanged (Table 1). Two methods of physical cell dispersion were investigated, (a) the classical revolving paddle method of Sayers et al. [10] and (b) repeated passage of adrenal tissue through teflon tubing attached to a syringe. In each of three paired experiments, cells were found to be more responsive when the latter method was used (Table 2).

Table 2. Comparison of two methods of cell dispersion Teflon tubing Cortisol (nmol/'l) Expt 1 Basal ACTH 10 9 M Expt 2 Basal ACTH 10 -8 M Expt 3 Basal ACTH 10-9M

Revolving paddle

Fold increase s

Cortisol (nmol/l)

Fold increase

1.7 25.9

14.9

1.9 5.3

2.7

2.7 7.4

2.7

3.1 5.4

1.8

7.5 110,6

14.8

8.2 65.6

8.0

~Fold increase = ACTH stimulated value/basal value.

Cell suspension protocol

In each experiment the adrenals from three guinea-pigs pre-treated as described above were used. Excess fat and connective tissue were removed and the adrenals were finely chopped. The tissue was incubated in Eagle's Modified Essential Medium (EMEM, Flow Labs, Scotland, 10-105-22) containing collagenase (Sigma, Poole, England, C0130) 2 mg/ml, for 20 min at 37°C. After centrifugation at 400g for 10min, the supernatant was discarded. The pellet was suspended in EMEM containing 0.2% bovine serum albumin (BSA). Cells were physically dispersed by passage up and down teflon tubing attached to a syringe. On standing, the dispersed cells remained in suspension; this suspension was then filtered through a nylon mesh. This process was repeated three times and the dispersed cells were combined and centrifuged as before. The resultant pellet of adrenal cells was washed in EMEM and centrifuged before resuspension in EMEM containing BSA 0.5g%, calcium 8 mM, and vitamin C 1 mM. Aliquots of the cell suspension, 800 #1, were added to 15 x 100mm plastic culture tubes which contained various POMC peptides in 100/~1 volumes. The following POMC-derived peptides were studied: ACTHj_24 (Ciba Labs, Horsham, West Sussex, England); 16K fragment, fl-LPH and 73-MSH (Peninsula Labs Europe, Merseyside, England); 8- and y-MSH, ~t-, fl-

Table 1. Effect of pre-treatment with ACTH a on cortisol and A4 production in isolated guinea-pig adrenal cells

Cortisol Basal Maximum response to ACTH A4 Basal Maximum response to ACTH

Untreated

Pre-treated

35 _+ 10 259 + 73

48 + 18 533 +_ 129"

4.3 +__1.7 16.8 _ 3.0

5.1 __. 1.1 50.9 + 11.5"*

Results are expressed as pmol/105 cells, mean + SE, n = 9. aPretreatment: single injection s.c. of ACTH (Acthar Gel) 3 days prior to adrenalectomy. *Significant increase, compared to untreated group, P < 0.05; **Significant increase, compared to untreated group, P < 0.01.

and ~-END, met- and leu-ENK (Sigma); JP, a gift from Dr L. N. Parker (University of California, Long Beach, CA). The adrenal enzyme inhibitor, trilostane 6/~M (Sterling Winthrop, England), was added to tubes in which dehydroepiandrosterone (DHEA) was to be measured. The final tube volume was made up to 1 ml with resuspending medium, where necessary. Each experimental condition was set up in duplicate and incubation was carried out under 100% oxygen. At the end of the incubation period the cell suspensions were stored frozen. Intracellular steroids were released into the incubation medium by repeated freezing and thawing. The contents of duplicate tubes were combined before steroid measurements were carried out. Steroid measurement

Cortisol in the incubation medium was measured by direct radioimmunoassay using antiserum from Guildhay (Guildford, Surrey, England), cortisol-3-(O-carboxymethyl)oximo(2-(125I))iodohistamine (Amersham Int., Amersham, England) and phosphate buffer 0.5 M, pH 7.4. Aliquots of cell lysates and standards were incubated in the presence of antibody and radiolabelled cortisol overnight at 4°C. Unbound cortisol was separated from antibody bound cortisol using 1% dextran-gelatincoated-charcoal, following centrifugation at 4000g for 15 min. The supernatant was decanted and the radioactivity in the free fraction (the charcoal pellet), was measured using a gamma counter. A4[ll] and DHEA[12] were measured by radioimmunoassay after extraction into diethyl ether and isolation of the steroids by chromatography over celite. A4 assay: radiolabel-A419-[125I]iodine derivative, Diagnostic Products U.K. Ltd, Abingdon, Oxfordshire, England; antiserum--Radioassay Systems Labs (RSL),

POMC peptides and adrenal steroidogenesis

79

Carson, U.S.A., Cat. No. 1440R1/P. DHEA Wilcoxin Rank Sum Test for non-parametric assay: radiolabel-dehydro[1,2,6,7- 3H]epiandro - data [15]. Values of P < 0.05 were considered to sterone, Amersham, Code TRK 157; anti- be significant. serum--RSL Cat. No. 1500 R1-46. The between-assay reproducibility estimated by RESULTS calculating the average of the coefficients of variation of 2-3 plasma pools each measured on Steroid response to P O M C peptides 7-15 occasions[13] was as follows: cortisol The steroids cortisol, A4 and DHEA re13.2%; A4 13.1%; and DHEA 13.9%. The sponded to ACTH stimulation in a dose depenwithin-assay reproducibility, evaluated from dent manner over the concentration range duplicate measurements of a plasma pool in 1 0 - 1 3 - 1 0 -9 M (Fig. 1). Stimulation was statisti7-15 assays[13], was as follows: cortisol cally significant (P < 0.01), in the presence of 9.4%; A4 9.7%; and DHEA 12.1%. The ACTH concentrations of I> 10-12 M. The maxisensitivities [14] of the radioimmunoassays mum stimulation achieved in the presence of were as follows: cortisol <98 fmol/tube; A4 ACTH was 12, 10 and 7 times basal levels for <7.8 fmol/tube; DHEA <31.1 fmol/tube. The cortisol, A4 and DHEA, respectively, fl-LPH specificity of the assays was assessed by submit- over a concentration range of 10-]°-10-8M ting known amounts of pure steroids to the stimulated steroid production (Fig. 2). Stimuentire assay procedure. The percentage cross- lation in the presence of fl-LPH 10-8M was 6, reactivity of potentially interfering steroids 5, and 5 times basal levels for cortisol, A4, and in the androgen assays were as follows: A4 DHEA, respectively. assay---cortisol, cortisone, corticosterone, 1lflThe steroid responses to the various POMC hydroxyandrosterone <0.25%, pregnenolone, peptides studied, at the top concentration used progesterone, 17-hydroxypregnenolone, 17-hy- in each case, are summarized in Table 3. The droxyprogesterone, etiocholanolone < 0.1%, N-terminal 16K fragment 10-SM stimulated DHEA, DHEA-sulphate <0.02%, testoster- steroid production, but only in the case of one, epiandrosterone, llfl-hydroxyandro- cortisol did this reach statistical significance stenedione <0.5%; DHEA assay--cortisol, (P < 0.05). JP 10 -7 M did not cause any significortisone, corticosterone, 17-hydroxypreg- cant change in steroid production, although nenolone, progesterone, testosterone, l lfl-hy- there was a trend towards stimulation of androdroxyandrosterone < 0.1%, pregnenolone, gen production. 73-MSH 10-6M and fl-MSH 17- hydroxyprogesterone, 1lfl -hydroxyandro- 10 -6 M both caused a slight increase in steroid stenedione <0.25%, epiandrosterone 0.43%, production, reaching significance (P < 0.05) for A4 0.06%, etiocholanolone, DHEA-sulphate cortisol. Three endorphins (~t-, fl- and 7-), were <0.02%. studied; these resulted in little or no change in steroidogenesis. Met-ENK 10-7M and IeuStatistical methods ENK 10 7 M did not stimulate steroidogenesis, The significance of differences between basal but in contrast resulted in a significant inhiand stimulated values were examined using the bition of DHEA production (P < 0.05). Cortisol

Androsteneclione

Dehydroepiondrosterone

~50C

n= T5

130 c

T10C 90C 70C 50C 3OO 100

TO-T3 |0"12 10-II 10-10 10-9 ACTH concentration, molar

10-13 10-12 ~O-tl 10-10 10-9 ACTH concentration, molar

10-~3 IO-IZ 10-11 10-9 ACTH concentration, molar

Fig. I. The cortisol, A4 and D H E A responses to ACTH 10 -'3 to 10-9M in isolated guinea-pig adrenal cells incubated for 2 h at 37°C. D H E A was measured in trilostane (6#M)-treated cells. Values are expressed as mean + SE relative to basal production of I00 where basal values were as follows: cortisol 47.1 + 13.2 pmol/10 ~ cells, n = 15; A4 4.3 + 1.0 pmol/105 cells, n = 15; DHEA 9.7 + 2.0 pmol/105 cells, n = 15. Significant increases as compared with basal production: *P < 0.05; **P < 0.01. SBMB 4 4 / I ~

80

Y v o N m O'CONNELL et al. Androstenedione

Oeh ydroe piond rosterone

10" ~ 104 2 x104 104t B-Upotropin concentration, molar

10-e 2 x l O -e I0 "e 6-Lipotropin concentration, molar

Cortisol 700 " 6OO 50O 4OO 3OO 20O too o

I0 "l° I0"e 2 x 1 0 -e I0"e B-Upotropin concentration, molar

Fig. 2. T h e cortisol A 4 a n d D H E A responses to f l - L P H 10-~°-10 -s M in isolated g u i n e a - p i g a d r e n a l cells i n c u b a t e d for 2 h at 37°C. D H E A was m e a s u r e d in trilostane ( 6 / ~ M ) - t r e a t e d cells. Values are expressed as m e a n + SE relative to basal p r o d u c t i o n o f 100 w h e r e basal values were as follows: cortisol 46.8 _ 12.0 pmol/105 cells, n = 11; A 4 4.4 + 1.2 p m o l / 1 0 s cells, n = 11; D H E A 1.5 _ 0.2 pmol/105 cells, n = 8. Significant increases as c o m p a r e d with basal p r o d u c t i o n : *P < 0.05; **P < 0.01.

Steroid response to P O M C peptides in the presence o f A C T H

The effects of various POMC peptides on cortisol and A4 production in the presence of ACTH is shown in Figs 3 and 4. fl-LPH 10 -s M significantly reduced the maximum response to ACTH 1 0 - 9 M of both cortisol (P < 0.05) and A4 (P < 0.01).//-END 10 -6 M significantly reduced the maximum cortisol response to ACTH 1 0 - 9 M (P < 0.01). a-END 10 -6 M significantly reduced the A4 response to ACTH 10-9M (P < 0.05). In the presence of a low concentration of ACTH, i.e. 10 -12 M, fl-LPH 10 -s M (P < 0.01), 16 K 10 -8 M (P < 0.05), fl-MSH 10 -6M (P <0.01) and Ieu-ENK 10 -6M (P < 0.05) significantly increased cortisol production. However, only in the presence of flLPH 10-SM and 16K 10-SM was the A4

response to ACTH 10-12M significantly increased.

DISCUSSION

The guinea-pig adrenal gland has been used as a model for the study of adrenal cortisol and androgen production for a number of reasons. The zonation of the guinea-pig adrenal cortex is similar to the human gland, having a particularly well-developed zona reticularis [8]. It has an active 17-hydroxylase enzyme system, required for cortisol and androgen synthesis [16] and cortisol is the major glucocorticoid product. Like the human adrenal, it has a requirement for ascorbic acid [9]. The guinea-pig adrenal cortex can synthesize considerable amounts of A418,16-18] and smaller amounts of

Table 3. Steroid production in response to POMC-derived peptides. Values expressed relative to basal = 100 Cortisol

ACTH 10-gM N-terminal pcptides 16K 10-SM JP 10-TM ~,3-MSH 10-6 M ~,-MSH 10-6M ~ ' - M S H 10 - 7 M

C-terminal peptides /LLPH 10-SM ~-MSH 10 -6 M ~-MSH

10-TM

~-END 10-6 M ~-END 10-7 M y-END 10-6 M a - E N D 10-6M a - E N D 10-TM Met-ENK 10-6M Met-ENK 10-7 M Lcu-ENK 10-6M Leu-ENK 10-7 M

A4

Mean

SE

n

1188"*

191

15

219" 106 116" 143

55 5 3 15

8 6 8 5

--

581"* 222*

DHEA SE

n

Mean

SE

n

153

15

725**

139

15

159 111 106 126 --

37 6 4 22

8 6 8

211 124 128

70 I1 36

6 6 6

28

5

92 69

11 7

183

8

13

7

10

10

4 19

4 5

97 I1

6 4

14

6

10

8

--

8

7

7

73*** -73***

7

7

1022"*

96 64

I1 7

477** 205

10

10

7 7

4 6

7

8

105 -91 114 -84 -95 --

--

121 -108 104 -97 -104 --

Mean

5

--

7

7

--

153

4

534** -117 -224 90 -93

cortisol value (Mean __.SE) 47. I +_. 13.2 pmol/I 05 cells, n ffi 15; basal A4 value 4.3 + 1.0 pmol/10 s cells, n = 15; basal DHEA value 9.7 + 2.0pmol/105 cells, n = 15. Significant stimulation vs basal, * P < 0.05; **P < 0.01; significant inhibition vs basal, ***P < 0.05.

Basal

P O M C peptides and adrenal steroidogenesis (A) Cortisol

i ooo

8O0 In

o 600

~u 4O0 n

200

'+

l

AOTH I0"12M

(B)

'" l'-" "l -" "l +'-" AOTH

I0-121LI

AOTH Io'U~I

AGTH IO-IIM

Androstenealone

r-~ 600 5oo

81

was not significantly altered (Table 1). It is possible that this priming with ACTH results in increased numbers of ACTH receptors in the cell membrane, which when activated result in greater responsiveness than in ceils which have not been primed. This report represents the first comprehensive study of the effects of POMC-derived peptides on androgen and cortisol production in vitro. Apart from ACTH the most significant stimulatory effects on steroidogenesis were seen in the presence of ]/-LPH. The results confirm and extend those of Pham-Huu-Trung et al. [IT], who reported that in 3 experiments /?-LPH 2.5-20 x 10-gM stimulated cortisol and A4 production in isolated guinea-pig adrenal cells. The present study also showed stimulation of (A)

400

* 1~

1600 "I

"~ 300 n

r"-I

Cortisol ..

NS

900

1:012~M

1~011"~U !

1:¢11~M

1,001~!M

Fig. 3. The steroid response to P O M C peptides in the presence of A C T H 10 -12 M. Cortisol and A4 were measured following incubation of isolated guinea-pig adrenal cells for 2 h at 37°C in the presence o f peptide alone t~; A C T H I0 -~2 M alone I-q; pcptide + A C T H 10 -12 M lB. Values are shown as mean + SE expressed relative to basal production of 100, where basal values were as follows: cortisol 47.1 + 13.2pmol/105 cells, n = 15; A4 4.3 + 1.0pmoi/10 s cells, n = 15. Significant increases as compared with production in the presence o f A C T H 10 -t2 M alone: *P < 0.05; **P < 0.01. NS, not significant.

DHEA [8, 19] and production of cortisol and A4 is responsive to ACTH [18]. In the present study A4 and DHEA were measured by radioimmunoassay following ether extraction and chromatography over celite columns. These assays are highly specific (see Methods) and it is therefore very unlikely that any known glucocorticoid, androgen or precursor steroid contributes significantly to the measured values. In optimizing the isolated guinea-pig adrenal cell system it was found that pre-treatment of guinea-pigs with a single injection of long-acting ACTH 3 days prior to adrenalectomy resulted in increased subsequent responsiveness to ACTH in vitro, while basal steroid production

~

1200

i

8oo

O. 40:

'+"t" t'°'l ACTH ACTH 10"414

(B)

l°-eM ~END pENO ÷ ACTH

t-t

10"4M *d[MD mEND AO+TH

Androstenedloae

1500 ]

NS

/

**

r--'l

1000

piP. ~ ,u..

/

AC1H AOTH

lo-*u

tttt

ACT. 104M llEND pE+ND AOTH

Aor. ~o"eM "JND dND + AOTH

Fig. 4. The steroid response to P O M C peptides in the presence o f A C T H 10- 9 M. Cortisol and A4 were measured following incubation of isolated guinea-pig adrenal cells for 2 h at 37°C in the presence o f peptide alone []; A C T H 10 -9 M alone I'-I; peptide + A C T H 10 -9 M II. Values are shown as mean + SE expressed relative to basal production o f 100, where basal values were as follows: cortisol 47.1 + 13.2pmol/105 cells, n = 15; A4 4.3 + 1.0pmol/10 ~ cells, n -- 15. Significant inhibition as compared with production in the presence of A C T H 10-gM: *P <0.05; **P < 0.01. NS, not significant.

82

YVONNEO'CONNELLet al.

D H E A production in the presence o f fl-LPH. There were previous reports o f stimulation o f aldosterone [20] and corticosterone [20, 21] production in the presence o f f l - L P H in isolated rat adrenal cell preparations. Significant stimulatory effects on steroidogenesis were seen in the presence o f 16K fragment 10 -8 M, 73-MSH 10 -6 M and fl-MSH 10 -6 M. There have been conflicting reports by other investigators o f the effects o f these P O M C derived peptides on steroid production. Pederson and Brownie [21] showed that 16K fragment stimulated corticosterone production in isolated rat adrenal cells, and that the active part o f the molecule was equivalent to the y3-MSH moiety. Lis e t al. [22] showed that the N-terminal fragment was a potent stimulator o f aldosterone production in isolated h u m a n adrenal cells. However, J o r n o t e t al. [20] showed no effect on corticosterone or aldosterone production in isolated rat adrenal cells in the presence o f 16K fragment, and Cathiard e t al. [23] demonstrated no change in steroidogenesis in the presence o f 73-MSH in ovine or bovine adrenal cells. The lack o f stimulatory effect in the presence o f the various endorphins studied is in keeping with studies using other model systems [17, 20, 21]. In isolated guinea-pig adrenal cells we observed no effect on cortisol or A4 production in the presence o f JP. There was a slight increase in the mean D H E A production levels in 6 experiments, but in two individual experiments no stimulation was seen. Parker e t al. [6] have postulated that JP is the specific cortical androgen-stimulating h o r m o n e since it was active by itself and synergistic with A C T H in stimulating D H E A secretion in isolated h u m a n adrenal cells, but had no effect on cortisoi secretion. However, others have been unable to confirm stimulation o f steroidogenesis by this P O M C sequence in cultured h u m a n adult[24] or foetal [25] adrenal cells. N o n e o f the P O M C derived peptides, including JP, examined in this study significantly favoured androgen over cortisol production. Investigation o f the effects o f POMC-derived peptides on ACTH-stimulated steroidogenesis revealed that f l - L P H inhibited maximally A C T H - s t i m u l a t e d androstenedione and cortisol production. In contrast, in the presence o f a low concentration o f A C T H , f l - L P H 1 0 - S M resuited in a significant increase in cortisol and A4 production. The agonist/antagonist effect o f flL P H suggests that it m a y be acting through the A C T H receptor.

Those POMC-derived peptides which stimulated steroidogenesis, fl-LPH, 16K fragment, y3-MSH and fl-MSH, all contain the heptapeptide sequence equivalent to ACTH,_t0. It would seem likely that the steroidogenic effects are consequent to their binding to an A C T H receptor. However, they are not equipotent, so the presence o f the heptapeptide p e r s e is not sufficient to explain their steroidogenic capacity. Further study o f their steroidogenic mechanism is warranted, particularly in the case o f fl-LPH. The present study suggests that fl-LPH m a y have a role in the modulation of adrenal steroidogenesis but provides no evidence to support a role for POMC-derived peptides in the specific control o f adrenal androgen production in guinea-pig cells. Acknowledgements--The authors wish to acknowledge the

generosity of Dr L. N. Parker who made "joining peptide" available for use in this project. This work was performed with the aid of a grant from the Health Research Board of Ireland. REFERENCES

1. McKenna T. J. and Cunningham S. K.: The control of adrenal androgen secretion. J. Endocr. 129 (1991) 1-3. 2. Parker L. N.: Adrenal Androgens in Clinical Medicine. Academic Press, New York (1989) p. 30. 3. Odell W. D. and Parker L. N.: Control of adrenal androgen production. Endocrine Rev. 10 (1985) 617-630. 4. Albertson B. D., Hobson W. C., Burnett B. S., Turner P., Clark R., Schiebinger R., Loriaux D. and Cutler G. Jr: Dissociation of cortisol and adrenal androgen secretion in the hypophysectomized adrenocorticotropin replaced chimpanzee. J. Clin. Endocr. Metab. 59 (1984) 13-18. 5. Genazzani A. R., Fachinetti F., Petraglia F., Pintor C., Bagnoli F., Puggiani R. and Carda R.: Correlations between plasma levels of opioid peptides and adrenal androgens in prepuberty and puberty. J. Steroid Biochem. 19 (1983) 891-895. 6. Parker L. N., Lifrak E., Shively J., Lee T., Kaplan B., Walker P., Calaycay J., Florsheim W, and Soon Schiong P. L.: Human adrenal gland cortical androgenstimulating hormone (CASH) is identical with a portion of the Joining Peptide of pituitary pro-opiomelanocortin. Endocrine Soc. 71st A. Meet. (1989) Abstr. 299. 7. Imura H.: Control of biosynthesis and secretion of ACTH: A review. Basic Res. Pharmac. Measur. 1 (1988) 1-6.

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