The hypothalamic-pituitary-adrenal axis

5 The Hypothalamic-Pituitary-Adrenal Axis COLIN M. FEEK DANIEL J. MARANTE CHRISTOPHER R. W. EDWARDS

In recent years there have been major advances in our understanding of the hypothalamic-pituitary-adrenal (HPA) axis, particularly in the field of secretion by anterior pituitary corticotrophin cells. The aim of this chapter is to review some of these developments and their possible clinical relevance.

BIOCHEMISTRY AND PHYSIOLOGY OF ACTH-RELATED PEPTIDES Anterior pituitary corticotrophin cells secrete adrenocorticotrophic hormone (ACTH) as part of a precursor molecule, pro-opiomelanocortin (POMe) (Mains, Eipper and Ling, 1977; Roberts and Herbert, 1977). Using recombinant DNA technology, Nakanishi et al (1979) have determined the nucleotide sequence for bovine POMC mRNA. The precursor protein consists of 265 amino acids encoding three peptides: ACTH, [3-lipotrophin ([3-LPH) and a 105 amino acid N-terminal sequence, N-POMC. The precursor is preceded by a 26-residue signal peptide (Figure 1). Pairs of dibasic amino acids flank each peptide and probably represent sites for enzymatic cleavage. The amino acid sequence His-Phe-Arg-Trp encoded within the Nterminal portion of the POMC is also shared by the melanotrophins a- and [3-MSH encoded within the peptides ACTH and [3-LPH respectively. The N-terminal MSH has been named y-MSH. The complete amino acid sequence of human N-POMC has more recently been determined (Seidah and Chretien, 1981). Human N-POMC consists of a peptide of 76 amino acids glycosylated at positions 45 and 65. The apparent molecular weight by gel filtration is 11200. The sequence NPOMC79 _109 which had not been accounted for during the isolation of NPOMC I 76 was later discovered as a 30-residue peptide ami dated at the Cterminus (Seidah et ai, 1981) and connects N-POMC I _76 to ACTH. Clinics in Endocrinology and Metabolism - Vol. 12, No .3, November 1983

597

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C. M. FEEK, D. J. MARANTE AND C. R. W. EDWARDS

The availability of purified N-POMC has made possible the production of specific radioimmunoassays (RIA) against different parts of the sequence (Hope et al, 1981). Smaller peptides containing the sequences of NPOMCSI _6 1 amide (y,-MSH), N-POMC SI 62 (Y2 -MSH) and N-POMC SI _76 (Yr MSH) have been synthesized and used in the production of RIA (Ling et ai, 1979).

SIGNAl PEPTIDE

N-TERMINAl FRAGMENT 11-76)

•

Y-MSH

---_... ACTH(1-391

a-MSH . (1-13)

ClIP (18-391

(J-LPHIH11

-

Y-lPH (1-58)

p-ENDOllPHIN 161-91)

(JMSH 141-58)

Figure 1. Structure of pro-opiornelanocortin (adapted from Nakanishi et ai, 1979, for bovine POMC); the connecting peptide N·POMC79_109 has been omitted .

The Gene for POMC The gene encoding human POMC has been located in the distal region of the short arm of chromosome 2, close to the gene for the enzyme acid phosphatase 1 (Owerbach et al, 1981). The gene for human POMC was isolated from a fetal DNA library and its nucleotide sequence was determined and compared with the DNA corresponding to bovine POMC mRNA. Three sequences are common to human and bovine POMC genes : (I) the sequence encoding ACTH; (2) the sequence encoding (3-LPH from position 36 to 89 which includes (3-MSH (sequence 39-56), Met-enkephalin (sequence 59-63) and (3-endorphin (sequence 59-89); (3) N-terminal POMC (sequence 1-76), In these three regions, the amino acid sequence is identical except for seven locations where there are one-base substitutions. There are two regions that differ: the connecting peptide that separates ACTH from N-POMC (NPOM~9_109)' and the segment immediately following ACTH «(3-LPH I _36 ) (Chang, Cochet and Cohen, 1980). Neurosecretion of POMC and Derived Peptides All three peptides derived from POMC are secreted concomitantly into the circulation and their concentration in plasma increases after adrenalectomy and becomes undetectable after hypophysectomy (Lis et al, 1982). Concomitant secretion of the three products has also been shown in cultures of mouse pituitary tumour cells; their secretion is stimulated by extracts of median eminence containing corticotrophin releasing factor (CRF) activity, and inhibited by dexamethasone (Oki et al, 1982). N-POMC I _76 is the main secretory form of the N-terminal peptide (Estivariz et al, 1980). It is secreted with ACTH and (3-LPH under a wide

HYPOTHALAMIC-PITUITARY -ADRENAL AXIS

599

range of physiological and pathological conditions. Hope et al (1981) observed a close correlation between plasma concentrations of N-POMC and ACTH reflecting co-ordinated synthesis and secretion in vivo. Studies with normal human and rat pituitary corticotrophs in vitro indicate that the three peptides are secreted in equimolar concentrations. This presumably reflects post-translational cleavage of the parent molecule, POMe. Biological Actions of POMC and its Products The species similarity of the three products of POMC suggests a related biological function. Although the biological actions of ACTH are well established, at least in regard to the adrenal gland, no definite function for [3-LPH and N-POMC has been determined. The observation that the first five residues of [3-endorphin are common with the opiate peptide Met-enkephalin led to the discovery of its potent opiate-like activity (Li, 1977). However, despite intensive research on the matter, no clear role for an adrenal or extraadrenal action of [3-LPH/[3-endorphin has emerged. The presence of POMC-derived peptides in extra-pituitary tissues has also been described, and a number of effects on learning, memory, analgesia and maintenance behaviour in experimental animals have been reported (for review, see De Wied and J olles, 1982). Actions of ACTH on the adrenal gland ACTH was known for many years before details of its precursor were elucidated. It is accepted at present that ACTH is the pituitary hormone responsible for the control of steroidogenesis by the adrenal cortex. A great deal of information regarding its structure, biological activity, structurefunction relationships, assays and mechanism of action has been published (for review, see Rees and Lowry, 1979). ACTH exerts many actions upon the adrenal cortex, including an increase in blood flow, production of hypertrophy, depletion of ascorbic acid, depletion of cholesterol esters and the synthesis of corticosteroids. It also stimulates the uptake of lipoprotein-bound cholesterol and induces a number of adrenal enzymes. The initial event in the action of ACTH is its binding to specific receptor sites on the mem brane of the adrenal cell. This is followed by the activation of the enzyme adenylate cyclase and the production of cAMP. Rapid increases in membrane phospholipids also occur (Farese, 1983). Actions of N-POMC on the adrenal gland Since the discovery of POMC there has naturally been a search for a possible biological role for the N-terminal peptide. Indeed, a 16K fragment corresponding to N-POMC, but derived from a mouse pituitary tumour cell line, has been shown to potentiate ACTH-induced steroidogenesis by isolated rat adrenocortical cells (Pedersen and Brownie, 1980). However it was necessary to trypsinize the 16K fragment for it to be effective, suggesting that biological

600

C. M. FEEK, D. J. MARANTE AND C. R. W. EDWARDS

activity resides in a smaller part of the fragment. This may be in the region of y-MSH. Synthetic YrMSH is also capable of potentiating ACTH stimulated adrenocortical steroidogenesis and to stimulate the activity of the adrenocortical enzyme cholesterol ester hydrolase (Pedersen, Brownie and Ling, 1980). Lys-YrMSH failed to stimulate an increase in cAMP or membrane phospholipids (Farese et ai, 1983). Binding sites for YrMSH have been demonstrated on isolated whole adrenal cells and in membrane fractions from adrenal glands as well as a number of other tissues (Pedersen and Brownie, 1983). However there was no evidence to suggest that there is any biological activity associated with the binding of the peptide to the cells and whether such binding is to a specific receptor. Nevertheless, potentiation by y-MSH is dose-dependent, producing a parallel shift of the dose-response curve of ACTH-stimulated adrenal steroidogenesis in hypophysectomized rats (Pedersen, Brownie and Ling, 1980). Other authors have also described the presence of ACTH-potentiating factors in high molecular weight peptides obtained from rat anterior pituitary extracts (lida et ai, 1981). Turning to other sources of N-POMC, human N-POMC significantly potentiates ACTH-stimulated steroidogenesis in vitro using perfused isolated rat adrenocortical cells (Al-Dujaili et al, 1981). The mechanism of action of potentiation remains to be elucidated, although it has been suggested that it may require the presence of intact transcriptional mechanisms, since the effect of N-POMC appears to be abolished by inhibitors such as actinomycin D and mithramycin (Al-Dujaili et al, 1982). However, other groups have failed to demonstrate a potentiation effect with human N-POMC when isolated guinea-pig adrenal cells were employed (Pham-Huu-Trung et ai, 1982). This discrepancy may reflect the methodological differences or differences in responsiveness related to different animal species. Porcine NPOMC has been shown to be a potent stimulator of human adrenal cells obtained from a patient with an aldosterone secreting tumour (Lis et al, 1981). It would appear that N-POMC or one of its derivatives may act as an amplifier of ACTH-induced adrenal steroidogenesis introducing the intriguing concept of prohormone amplification. A further and interesting role has been suggested for N-POMC-derived peptides (Estivariz et al, 1982). Some fragments (N-POMC l _28 ) derived by limited proteolysis from N-POMC are potent stimulators of adrenal DNA synthesis in vitro, and of adrenal mitosis in vivo. Furthermore, the administration in vivo of antisera directed against the extreme N-terminal portion of N-POMC I _28 and the mid-portion of N-POMC I _74 both prevented compensatory hyperplasia of the adrenal gland remaining after unilateral adrenalectomy (Silas, Linton and Lowry, 1983). Thus N-POMC may be responsible, in part, for the regulation of adrenocortical cell growth. The activation of cholesterol ester hydrolase activity, originally observed by Pedersen and Brownie (1980) is also mediated by another region within N-POMC, N -POMC 57 _76 . Actions of fJ-LPH on the adrenal gland Little is known about the adrenal action of fJ-LPH/fJ-endorphin. However, it has long been thought that a non-ACTH pituitary factor may playa role in the regulation of aldosterone during sodium depletion. Indeed fJ-LPH has been

HYPOTHALAMIC-PITUITARY-ADRENAL AXIS

601

shown to stimulate aldosterone secretion by isolated rat adrenal zona glomerulosa cells (Matsuoka et al, 1981). {J-MSH was equally potent as {JLPH but concentrations of the peptides were pharmacological rather than physiological. {J-Endorphin failed to stimulate aldosterone secretion, unlike previous reports (Shanker and Sharma, 1979). Sodium depletion enhances the sensitivity of adrenal zona glomerulosa cells to {J-MSH (Yamakado, Franco-Saenz and Mulrow, 1982) and aldosterone concentrations obtained by maximal doses of {J-MSH were similar to those achieved by maximal doses of angiotensin II. Thus (3-MSH and related peptides may playa role in the regulation of aldosterone secretion in the rat, particularly in the salt-depleted state. Neuroregulation of ACT" Secretion Anterior pituitary corticotroph cells receive blood from the hypothalamicpituitary portal circulation. Secretion of POMC is under the direct control of the hypothalamic neurosecretory hormone known as corticotrophin releasing factor (CRF). This peptide is secreted into the portal circulation by neurones whose somas are localized in the mediobasal hypothalamus, with axons projecting into the median eminence. CRF is secreted in the vicinity of the portal capillaries in an episodic manner. There are three major factors involved in the secretion of ACTH:

Circadian rhythm. The existence of a circadian periodicity of plasma corticosteroid levels is well documented in normal human subjects and other species. Peak levels of ACTH occur prior to or at the time of awakening and decline during the day, reaching a nadir during the late evening/early morning hours when episodic peaks of secretion are scarce or absent. This circadian rhythm in the human is independent of posture, persists with bed rest and persists in the absence of the adrenals, indicating an endogenous periodicity, synchronized by environmental phenomena such as the light-dark cycle (Krieger, 1978). Negative feedback. Corticosteroids exert negative feedback upon ACTH secretion. Two components of this feedback have been described: short delay, rate-sensitive, associated with the rate of rise of the plasma corticosteroid concentrations and possibly mediated by an effect on the release of CRF from the hypothalamus, and delayed feedback, level-sensitive, associated with the concentration of steroid reached in plasma, and probably mediated by an influence of corticosteroids on the rate of synthesis of CRF and/or the sensitivity of the corticotrophs to CRF (Jones, 1978). Stress. Negative feedback of corticosteroids upon the hypothalamus and pituitary can be overridden by severe stress such as burns, surgery or hypoglycaemia to secrete ACTH, and markedly elevated levels of both corticosteroids and ACTH are found in the circulation at these times (Nelson, 1980).

602

C. M . FEEK, D . J . MARANTE AND C. R. W . EDWARDS

Identity and biological activity of CRF Although CRF was the first hypothalamic hormone to be demonstrated, attempts to isolate it in pure state were unsuccessful. One of the complicating factors has been the presence in posterior pituitary and median eminence of vasopressin (VP). This neurohypophyseal hormone per se has CRF-like activity. Recently, a 41-amino acid polypeptide has been isolated from ovine hypothalamus. It is highl y potent in stimulating secretion of ACTH by cultured anterior pituitary cells, and synthetic peptides with similar structure display CRF acti vity in vitro and in vivo (Vale et al, 1981). Immunoneutralization in vivo by exogenous anti -CRF antiserum inhibits the ACTH response to exogenous CRF and stress, and markedly inhibits ACTH levels in adrenalectomized rats (Rivier, Rivier and Vale, 1982). Moreover, the activity of the putative 41-residue CRF in vitro is potentiated by arginine-VP (Turkelson et al, 1982). However, the stimulation of CRF by stress seems to be independent of the potentiating effect of VP, as antagonists of the neurohypophyseal nonapeptide do not abolish ACTH response to psychological stress (Mormede, 1983). It is thus likely that VP may be a biological cofactor of CRF for certain types of response only. Neuroregulation of CRF secretion The activity of the CRF-producing cells is modulated by the nerve inputs from other areas of the brain (for review see Weiner and Ganong, 1978). Noradrenergic nerves are possibly involved in a negative control of CRF secretion (Lancranjan, Ohnhaus and Girard , 1979). In vivo and in vitro studies support the view that serotoninergic neurones stimulate the release of CRF and may play a part in the diurnal rhythmicity of adrenocortical function (Fuller, 1981). A role for histamine in the control ofCRF secretion is more controversial, and part of the uncertainty derives from the fact that it is unclear whether the amine is located in nerve cell terminals or in mast cells close to the vessels of the median eminence. It is recognized that systemic administration of histamine stimulates ACTH secretion: agonists of HI receptors administered directly to the third ventricle stimulate the secretion of ACTH and vasopressin. y-Aminobutyric acid (GABA) may have an inhibitory action on CRF secretion, and this has been used in the treatment of patients with Nelson's syndrome with drug agonists of GAB A (Dornhorst et aI, 1983). Opiate peptides are also possible modulators of CRF secretion; the opiate agonist peptide D-Ala2-MePhe4-Met-enkephalin-O-ol (DAMME) inhibits the secretion of ACTH (Delitala, Grossman and Besser, 1981), and naloxone causes an elevation in serum cortisol in normal human subjects, suggesting that a tonic inhibition on ACTH is exerted by endogenous opiates (Grossman and Clement-Jones, 1983). The existence of a brain renin-angiotensin system has attracted much interest since the preliminary reports by Fischer-Ferraro et al (1971) and Ganten et al (1971), and a number of actions have been described for centrally administered angiotensin II, including elevations of blood pressure, thirst and stimulation of VP release (Phillips, 1969). ACTH secretion is also

HYPOTHALAMIC-PITUITARY-ADRENAL AXIS

603

elicited by centrally applied angiotensin II but this effect may be due either to the actions of vasopressin or to a direct action of the polypeptide on anterior pituitary cells (Mangiapane and Simpson, 1980).

CLINICAL ASPECTS OF THE HPA AXIS The steroids secreted by the adrenal cortex are principally glucocorticoid, mineralocorticoid and sex steroids. The adrenal cortex is divided anatomically into three zones-glomerulosa, fasciculata and reticularis-but such a zonation is not necessarily functional. Whilst it has been established that glomerulosa cells secrete aldosterone and not cortisol, there is no evidence that sex steroids are secreted by a distinct morphological zone. Adrenal steroid secretion is generally regulated by ACTH but the major stimulus to the secretion of aldosterone is the renin-angiotensin system. A factor other than ACTH has been suggested in the control of adrenal androgen production. Evidence for this is far from convincing. In this section clinical disturbances of the HPA axis will be discussed in relation to function of the target organ-the adrenal gland-with particular reference to the three major groups of steroids secreted. Much has been reviewed elsewhere (James, 1979) and we will concentrate on some of the more recent developments in the field. Adrenocortical Glucocorticoids Cushing's syndrome The clinical and biochemical features of Cushing's syndrome are now well described and result from the sustained and inappropriate elevation of free plasma cortisol. The causes are illustrated in Table 1. Investigation of a suspected patient requires firstly that the patient has inappropriate hypercortisolaemia and secondly that the cause of the syndrome be determined (Table 2). Quite clearly one of the keys to the diagnosis of the cause of Cushing's syndrome is the 0900 plasma ACTH concentration (Rees, 1977). Whilst it is well recognized that care must be taken in the collection of the specimens it is also apparent that the timing of the sample is critical if meaningful results are to be obtained. Morning plasma concentrations of ACTH in patients with a corticotrophinoma are commonly believed to overlap the normal range. However normal ranges are often derived from blood samples taken between 0800 and 0900 or 1000 hours. While plasma ACTH concentrations may vary from 9 to 77 ng/l when sampled between 0600 and 0930, the range for 0900-0930 plasma ACTH is much smaller at 9 to 24 ng/l (Horrocks and London, 1982). In eight patients with corticotrophinoma 0900-0930 plasma ACTH concentrations were obviously elevated at 39 to 109 ng/l when compared with the redefined normal range. ACTH secretion by corticotrophinomas is relatively autonomous at a level that is commonly found in the early-morning surge of normal individuals and such patients will only be seen to have raised levels if sampled later in

604

C. M . FE EK, D. J. MARANTE AND C. R. W. EDWARDS

the day when the surge in normal individuals should have subsided (Figure 2). Nevertheless, plasma ACTH estimation by radioimmunoassay does not necessarily reflect its biological activity. Table 3 demonstrates dissociation between cytochemical and radioimmunoassay estimations of ACTH in a patient with a proven corticotrophinoma. Chromatography of

Table 1. Causes of Cushing's syndrome ACTH dependent Iatrogenic Pituitary corticotrophinoma Ectopic ACTH independent Iatrogenic Alcohol induced Adrenal tumour (adenoma, carcinoma)

the pituitary tumour extract, following surgery, revealed the secretion of 'big' ACTH, N-POMC and f3-LPH (Figure 3) so that it is possible that loss of post-translational cleavage resulted in loss of prohormone amplification and lower biological activity. Indeed the patient had mild Cushing's disease, clinically and biochemically, despite the grossly elevated plasma ACTH concentrations estimated by radioimmunoassay. The tumour proved to be locally aggressive. Table 2. Inv estigation of suspected Cush ing's sy ndrome Does the patient have Cushing's syndrome? Circadian rhythm of plasma cortisol Suppression with low dose dexamethasone (2 mg /day) Urinary free corti sol excretion Insulin tolerance test What is the cause of the Cu shing's syndrome? 0900 plasma ACTH Metyrapone test Suppression with high do se dexamethasone (8 rug/day)

The secretory pattern of POMC products of human pituitaries obtained from patients with Cushing's disease, Nelson's syndrome and ectopic ACTH-producing tumours has been determined in an effort to demonstrate any abnormality in the mode of processing of the precursor by neoplastic cells. Dispersed pituitary cells obtained from tumour specimens removed by trans-sphenoidal surgery from patients with Cushing's disease were perfused in vitro (Oki et al, 1981). The gel filtration elution pattern of the the secreted peptides is consistent with the production of the three main

605

HYPOTHALAMIC -PITUITARY-ADRENAL AXIS

peptides derived from POMC in normal pituitary cells (i.e., ACTH, f3-LPH/ f3-endorphin and N-POMC). Furthermore, the addition of lysine vasopressin (LVP) and rat median eminence extracts to the perfusion system enhanced the release of the three peptides. Thyrotrophin-releasing hormone (TRH) and luteinizing hormone-releasing hormone (LHRH) also elicited the concomitant release of these peptides by the cells of one patient in whom combined administration of TRH and LHRH significantly augmented plasma cortisol levels in vivo when studied preoperatively. Fragments of a pituitary adenoma from a patient with Cushing's disease were maintained in culture for four days. Gel chromatography and radioimmunoassay of the culture medium revealed the presence of the three main products of POMC as well as minimal amounts of y-MSH and YI-

T

-80 Cushings

-70

0900-0930

-60

Plasma ACTH (ng/O

-50 -40

Normal

0830-0900

-30 -20

r

Normal

0900-0930

-70

'----'---'------'

0800 0830 0900 0930 TIME Figure 2. Plasma ACTH concentrations between 0830-0900 hand 0900-0930 h in normal individuals when compared with those concentrations between 0900-0930 h from patients with a corticotrophinoma (adapted from Horrocks and London, 1982).

606

C. M. FEEK. D. J . MARANTE AND C. R. W. EDWA RDS

MSH (Shibasaki et ai, 1981). Short-term cultures of fragments of a pituitary adenoma removed from a patient with Nelson's syndrome were also shown to secrete high molecular weight forms of N-POMC as well as ACTH and (3LPH. The secretion of the three peptides was stimulated by arginine vasopressin (A VP), TRH, substance P and Met-enkephalin, and suppressed by somatostatin and Leu-enkephalin, confirming that the abnormal sensitivity to some neuropeptides observed in vivo may be due to the loss of specificity of the receptor of the tumour cells, or more likely to a derepression phenomenon resulting in the appearance of multiple receptors that are not expressed in the membrane of normal corticotrophs . (Shibasaki and Masui, 1982). Table 3. Plasma ACTH concentrations as measured by radioimmuno-

assay and cytochemical bioassay in a female with a corticotrophinoma ACTH ng/l by: I . Radioimmunoassay 2. Cytochemical assay fJ-LPH ng/l

0900 ISOO 2400 0900

256 254 IS3 23

(10 to SO)

0900 ISOO 2400

2400 1135

(25 to 2(0)

« 10)

996

Analogous studies performed with tissue removed from patients with ectopic ACTH-secreting tumours have shown the secretion of smaller molecular species of y-MSH-like activity in addition to the three main products of POMC (Tanaka et al, 1981). The elution pattern on chromatography revealed some abnormalities in the glycosylation of y-MSH in some ectopic ACTH-producing tumours (Tanaka et al , 1981). This finding supports the view that the carbohydrate moieties attached to the POMC peptide may confer specific conformational properties upon the molecule so as to protect it from random proteolysis, and direct its processing by limited proteolysis. The role of ACTH in the cause of alcohol-induced pseudo-Cushing's syndrome has been conflicting . Estimation of plasma ACTH concentrations in these patients has been variable. Chronic alcoholism may occasionally result in unequivocal clinical and biochemical features of Cushing's syndrome that disappear over a few days following alcohol withdrawal. In these patients the half-life of cortisol is prolonged increasing free cortisol but the total cortisol is often low due to low circulating corticosteroid-binding globulin (CBG) levels. The increase in free cortisol exerts negative feedback at the level of the anterior pituitary so that the frequency and amplitude of cortisol secretion becomes depressed (Rosman et al, 1982). The treatment of Cushing's syndrome requires accurate diagnosis of the cause so that appropriate therapy can be directed towards the cause. Transsphenoidal microsurgery has revolutionized the treatment of corticotrophinomas and is the treatment of choice provided there is access to a

607

HYPOTHALAMIC-PITUITARY-ADRENAL AXIS

skilled neurosurgeon. Conventional treatment with bilateral adrenalectomy and corticosteroid replacement has disadvantages. The operation itself has a reported mortality of about 4 per cent. Cortisol hypersecretion may recur despite 'total' bilateral adrenalectomy because continued ACTH secretion may stimulate the production of corticosteroid either from remnant adrenal tissue or accessory glands (Chalmers, Mashiter and Joplin, 1981). There is /3-endorphin

!

.

v.v. 50

30K

~

22K

+

/3LPH

t

'YLPH

t

1-39ACTH

+

25

0 500 'C'LPH

250

0 ng/ml

500 'N'LPH 250

0 500

250

0 50

60

70

80

100

90

110

120

130

140

ml

Figure 3. Chromatographic profiles on Sephadex 0-75 of an extract of a corticotrophinoma from a patient (V.P.) with Cushing's disease in whom there was a dissociation in the plasma concentrations ofimmunoactive and biologically active ACTH.

608

C. M. FEE K, D. J . MARANT E A ND C. R. W. EDWARDS

an increase in radiologically abnormal pituitary fossae following bilateral adrenalectomy, rising from 19 to 44 per cent as a result of corticotrophin stimulation by loss of negative feedback inhibition (Besser et aI, 1972). An expanding corticotrophinoma, following bilateral adrenalectomy, causes pigmentation and often eventual visual pathway compression - Nelson's syndrome. This can usually be prevented by external pituitary irradiation at the time of adrenalectomy. External pituitary irradiation has been employed as primary treatment but cures only 10 to 20 per cent of adults, although it is highly effective in children. Medical treatment for Cushing's disease may help to ameliorate the condition but is not a definitive cure. Metyrapone can be used to prepare the patient for surgery (or as an adjunct to pituitary irradiation). Aminoglutethimide and trilostane are alternative competitive enzyme blocking drugs. The former has unacceptable sideeffects and the latter is of unproven efficacy. Cyproheptadine, a serotonin antagonist, and bromocriptine have occasionally been employed to suppress ACTH secretion. Clearly the aim of treatment is selective destruction of the tumour with preservation of surrounding normal anterior pituitary tissue . Transsphenoidal microsurgery can achieve this with normalization of plasma cortisol concentrations in 80 per cent of cases. In a large series (Laws et aI, 1982) of 65 patients operated upon between 1974 and 1980 a tumour was identified and removed in 62. In the remaining three, anterior hypophysectomy was attempted in two and a colloid cyst removed in one. The mean period of follow up was 20 (range 1 to 66) months. Following surgery there is usually a period of prolonged cortisol and ACTH deficiency until the function of suppressed corticotrophin tissue surrounding the tumour returns to normal. Recurrence of the disease in the short term is uncommon and is presumably due to residual tumour in inaccessible areas such as invasion into the cavernous sinus. However long term follow up is crucial to determine whether these tumours arise de novo from the anterior pituitary or as a result of abnormal control by the hypothalamus. Adrenocortical insufficiency In most centres primary adrenocortical insufficiency is diagnosed on the basis of clinical and biochemical features and the failure of the plasma cortisol to respond to a pharmacological dose of ACTH (250 /-lg tetracosactrin). Estimation of the 0900 plasma ACTH is an alternative but time should not be wasted by delaying treatment of the patient until the result of the plasma ACTH becomes available. The advantages of the estimation of plasma ACTH concentrations in patients with primary adrenal insufficiency is that it will identify a few patients with Addison 's disease who respond normally to tetracosactrin. It can also be used to monitor replacement therapy and assist in the differential diagnosis of secondary adrenocortical insufficiency. It is now possible to measure plasma (3-LPH. This has the advantage of reflecting plasma ACTH concentrations without the inherent instability. Plasma samples can even be separated and sent by post. Plasma cortisol profiles have long been used to monitor glucocorticoid replacement in patients with Addison's disease but have the disadvantage of

609

HYPOTHALAMIC-PITUITARY-ADRENAL AXIS

estimating total and not free cortisol. Plasma ACTH profiles have therefore also been employed to assess the adequacy of glucocorticoid replacement (Feek et al, 1981). With conventional replacement therapy (either hydrocortisone 30 mg or cortisone acetate 37.5 mg per day) approximately onethird of patients have suppressed ACTH secretion indicating over-replacement, one-third have elevated ACTH concentrations which suppress adequately with replacement therapy and one-third have elevated ACTH concentrations which fail to suppress, suggesting under-replacement (Figure 4). In this latter group plasma 0900 ACTH concentrations failed to suppress sufficiently in response to the oral administration of pharmacological doses

-

1()()()

1000

900

900

800

800

~700 E c

:::600 o

Vl

;: 500

~

u

«

~

400 ~

400

:>': Vl

5300

D.-

200

200

100

100

...---- ....---.

8

.-:I

0

TIME (hours)

Figure 4. The relation of plasma cortisol (left) to the degree of suppressibility of plasma ACTH concentrations (right) in twelve patients with primary adrenocortical failure following the oral administration of hydrocortisone 20 mg. Mean ± s.e. plasma cortisol concentrations were not significantly different when the group with suppressible plasma ACTH concentrations (e---e, n = 3) were compared with non-suppressible (x--x, n = 4) and suppressed (e--e, n = 5) plasma ACTH concentrations. From Feek et al (1981) with kind permission of the editor of Clinical Endocrinology.

of dexamethasone, indicating a degree of autonomy (Table 4). Whether it is justified to consider diminished sensitivity of the feedback mechanism as an indication of corticotroph hyperplasia is open to question. Certainly, sellar enlargement can be demonstrated in patients with Addison's disease but the generation of a 'feedback tumour' is probably a rare condition (Himsworth, Lewis and Rees, 1978). Primary adrenocortical insufficiency may present in childhood as a familial syndrome associated with tall stature (familial glucocorticoid insufficiency) (Thistlethwaite et al, 1975). Such patients have elevated plasma ACTH concentrations, subnormal plasma cortisol concentrations and a poor cortisol response to ACTH. Clinically there are symptoms and

610

C. M. FEEK, D. J. MARANTE AND C. R. W. EDWARDS

signs attributable to glucocorticoid rather than mineralocorticoid deficiency. Despite subnormal concentrations of aldosterone, plasma renin activity is normal, suggesting that another corticosteroid is exerting a mineralocorticoid effect. Indeed plasma concentrations of deoxycorticosterone have been shown to be elevated in these patients. The nature of the defect therefore remains obscure but the condition is easily treated with hydrocortisone replacement. Table 4. Plasma ACTH concentrations at 0900 h immediately before starting dexamethasone (day I) and on each of the succeeding days (days 2 to 5) in 3 patients with Addison's disease

Plasma ACTH (ng/I)

Patient

570 322 1812

I

2 3 Day of dexamethasone Dose of dexamethasone (rug/day)

I

2

224 206 37

189 293 66

51 48 60

75 45 30

2 2

3 8

4 8

5

Adrenocortical Mineralocorticoids Aldosteronism Primary hyperaldosteronism is usually thought to be an uncommon cause of hypertension accounting for less than 1 per cent of unselected hypertensives (Ferriss et al, 1981). The pathogenesis of this condition is usually due to an adrenocortical adenoma or idiopathic hyperplasia. Aldosterone producing tumours are sensitive to ACTH but not angiotensin II, so that when a patient with this condition stands upright there is no rise in plasma aldosterone concentrations. The plasma aldosterone then follows the normal ACTH-induced circadian rhythm (Figure 5). In idiopathic hyperplasia, the adrenal cortex is highly sensitive to angiotensin II, resulting in an excess secretion of aldosterone with a grossly exaggerated response on standing (despite only a small increase in plasma renin activity). Although ACTH and the HPA axis have little to do with the pathogenesis of these conditions, our understanding of the interaction of ACTH and angiotensin II upon glomerulosa cells has enabled us to devise these provocative tests to help in diagnosis. Nevertheless, there does appear to be a rare subgroup of patients with idiopathic hyperplasia in which ACTH may have a fundamental role in its pathophysiology. This condition is often familial, has the clinical and biochemical features of aldosteronism but can be ameliorated by the oral administration of glucocorticoid (glucocorticoidsuppressible hyperaldosteronism, aSH). Patients with aSH show an anomalous fall in plasma aldosterone on standing, in similar fashion to those patients with tumour and in marked contrast to the remaining patients with hyperplasia. Suppression of the HPA axis with the oral administration of glucocorticoid achieves a reduced plasma aldosterone concentration which then responds normally to changes in posture (see Figure 5). It would

611

HYPOTHALAMIC-PITUITAR Y-A DRENA L AXIS

therefore appear that in GSH there is an abnormal response of glomerulosa cells to chronic ACTH stimulation causing hyperplasia, aldosteronism and suppression of the renin-angiotensin system. This abolishes the postural response. Suppression of the ACTH drive with the oral administration of glucocorticoid reduces plasma aldosterone concentrations regaining normal renin-angiotensin system function (Ganguly, Grim and Weinberger, 1981). The intriguing question is the cause of the abnormal response of glomerulosa cells to ACTH. Almost certainly the fault resides at the level of the glomerulosa cell and is not due to the pituitary secreting an abnormal prohormone with increased prohormone amplification.

Hyperplasia

plasma aldosterone

<:

Erect/

<.

Tumou~

......... . .

Supine--··-, -,

0900

"-'

..... 1200

Normal

X

GSH on

glucocorticoid

0900 0.....0

/200

X

Tumour+ Hyperplasia

0900

/200

X GSH

Figure 5. Postural responses of plasma aldosterone concentrations. In normal individuals plasma aldosterone concentrations follow the normal circadian rhythm if the individual remains recumbent (e- - -e) but rise on standing (e- - (» when the renin-angiotensin system is activated . Patients with an aldosterone secreting adrenocortical tumour achieve plasma aldosterone concentrations that fail to respond to changes in posture and mimic the natural circadian rhythm . Patients with hyperplasia achieve an exaggerated response to posture. A rare subgroup of patients with hyperplasia (GSH) behave in a similar manner to a tumour but correct to normal with the administration of glucocorticoid.

Another syndrome is that of apparent mineralocorticoid excess characterized by low renin hypertension, hypokalaemia, subnormal production of known mineralocorticoids, a defect in the peripheral metabolism of cortisol and a decrease in blood pressure in response to spironolactone administration. ACTH and cortisol concentrations are suppressed and the exogenous administration of either of these increases blood pressure and decreases serum potassium. There have been two interpretations of this syndrome. The first is that a genetic defect in cortisol-l l-oxidase results in secretion of bioactive cortisol (no conversion to cortisone), with suppression

612

C. M. FEEK, D . J . MARANT E AND C. R. W . EDWARDS

of ACTH and decreased secretion of other corticosteroids. Alternatively, there may be a defective mineralocorticoid receptor which has an abnormal affinity for cortisol and thus may be genetically linked to the l l-oxidase defect. Cortisol thus becomes the major mineralocorticoid as well as glucocorticoid (New et ai, 1982). Adrenocortical Sex Steroids Adrenarche Sex steroids are seen in high concentrations in the blood of the newly born but are soon suppressed and remain so until puberty emerges. The adrenal secretion of androgens, dehydroepiandrosterone (DHA), its sulphate (DHA-S04 ) and androstenedione increases significantly prior to the onset of puberty (adrenarche) (for review see Ducharme and Collu, 1982), The prepubertal and partly pubertal growth spurt, with the development of axillary and pubic hair, results from the adrenarche. These adrenal androgens are principally under the control of ACTH and can be suppressed by dexamethasone. Nevertheless some authors have felt it necessary to invoke a further hypophyseal hormone, cortical androgenstimulating hormone (ASH), capable of stimulating adrenal androgen production (Parker and Odell, 1979). The relationship of the adrenarche to

400

300

ACTH ng/ml

200

100

(a)

9

11

13

15 17 19 Clock Time

21

23

613

HYPOTHALAMIC-PITUITARY-ADRENAL AXIS

gonadarche is debatable. It is possible that adrenal androgen secretion initiates activation of the hypothalamic gonadal axis and the gonadarche. In support of this is the finding that adrenal androgens are elevated in the plasma of patients with idiopathic hypogonadism (Cohen et ai, 1981). However, the adrninstration of DHA-S04 to a male patient with familial cytomegalic adrenocortical hypoplasia failed to induce pubertal development (Cohen et ai, 1982). Nevertheless, treatment with DHA-S04 did increase plasma testosterone concentrations indicating, perhaps, an important link between adrenal cortex and gonad. It is also possible that the 'adrenarche' and 'gonadarche' occur in parallel and independently of each other. Congenital adrenal hyperplasia Thi s condition has recently been well reviewed in this series (Hughes, 1982). The main problem in the long term is to suppress the HPA axis with exogenously administered corticosteroid in order to suppress excess adrenal androgen production to allow adequate growth, pubertal development and in females menstruation and fertility. The optimal steroid regimen remains controversial but the ideal treatment should suppress the HPA axis without

800 700

600 17-0H 500 Progesterone nmol/l 400

300

200 100 (b)

9

11

13

15 17 Clock Time

19

21

23

Figure 6. (a) Pla sma ACTH and (b) 17aOH-progesterone concentrations in four patients with congenital and adrenal hyperplasia before (- - -) and after (--) treatment with oral de xamethasone 0 .5 mg on retiring and prednisolone 2.5 mg on waking . Two patients ( ,~~ . e) previously recei ved oral prednisolone 7.5 mg /day and two ( x , [ ;j received cortisone acetate 20 mg/day in di vided do ses.

614

C. M . FEEK, D. J . MARANTE AND C. R. W. EDWARDS

producing symptoms and signs of glucocorticoid excess. Often hydrocortisone in doses of 10 to 20 mg/mz -day in three divided doses is recommended but small doses of longer acting synthetic glucocorticoids given at night may provide better control of precursor secretion (Hayek, Crawford and Bode, 1971; Smith et al, 1980). It is easier to suppress the HPA axis with the adminstration of glucocorticoid at night and it is our practice to divide the daily dose of glucocorticoid in a pattern of reverse circadian rhythm. Usually we give prednisolone 2.5 mg orally on waking and dexamethasone 0.5 mg on retiring at night (Figure 6). This regimen usually achieves adequate suppression of ACTH concentrations but it must be remembered that patients with CAH may have a degree of corticotroph hyperplasia (Table 5). Suppression of the HPA axis may therefore require a temporary period of glucocorticoid over-replacement prior to a period of maintenance on lower dose. Glucocorticoid therapy may be monitored by 24-hour profiles of ACTH, cortisol, 17aOH-progesterone and mineralocorticoid therapy by plasma renin concentrations. In females the most discriminating monitor is the development of normal menstruation and fertility. The need for such strict control in postpubertal males, however, is debatable. There is the possibility that such patients may develop a 'feedback' tumour if poorly controlled.

Table 5. Plasma ACTH con centrations at 0900 h immediately before starting dexamethasone (day J) and on each of the succeeding days (days 2 to 5) in a patient with congenital adrenal hyperplasia

Plasma ACTH (ng /I) Day of dexamethasone Dose of dexamethasone (mg/day)

120 I

2

48 2 2

50 3 8

45 4 8

52 5

SUMMARY

Anterior pituitary corticotrophin cells secrete ACTH as part of a larger precursor molecule, pro-opiornelanocortin. Post-translational cleavage of this precursor yields three major peptides: ACTH, {3-LPH and N-POMC. Experiments both in vivo and in vitro suggest that N-POMC may act as a prohormone amplifier for ACTH-induced adrenal steroidogenesis and as regulator of adrenocortical cell growth. The secretion of POMC is under the control of CRF. These findings are discussed in relation to the pathophysiology of corticotrophinoma. The primary defect in this condition appears to reside at the level of the anterior pituitary cell and is readily amenable to treatment by trans-sphenoidal microsurgery. The estimation of plasma ACTH concentrations is proving useful in the monitoring of various clinical conditions including Addison's disease and congenital adrenal hyperplasia.

HYPOTHALAMIC-PITUITARY-ADRENAL AXIS

615

ACKN OW LEDGEMENTS

We would like 10 thank Ms Alison Munro for her invaluable help and Dr Brent Williams for his expert advice. We would also like to thank Professor Lesley H. Rees for the preparation of Figure 3.

REFERENCES Al-Dujaili, E. A. S., Hope, J., Estivariz, F. E . et al (1981) Circulating human pituitary proy-melanotropin enhances the adrenal response to ACTH . Nature, 291, 156-159. Al-Dujaili, E. A . S., Williams, B. c., Edwards, C. R. W. et al (1982) Human y-melanotropin precursor potentiates corticotropin-induced adrenal steroidogenesis by stimulating mRNA synthesis. Biochemical Journal, 204,301 -305. Besser, G. M. , Ratcliffe, J. G., Cryer, R. J. & Scott , A. P. (1972) Total adrenalectomy and its effect on plasma ACTH and pituitary function in pituitary dependent Cushing 's syndrome. In Cushing's Syndrome (Ed.) Binder, C. & Hall, P . E . pp. 132-140. London: Heinemann. Chalmers, R. A., Mashiter, K. & Joplin, G. F. (1981) Residual adrenocortical function after bilateral 'total' adrenalectomy for Cushing's disease. Lancet, ii, 1196-1199. Chang, A. C. Y., Cochet, M. & Cohen, S. N . (1980) Structural organization of human genomic DNA encoding the pro-opiornelanocortin peptide. Proceedings of the National Academy ofSciences (USA), 77,4890-4894. Cohen, H., Wallace, A . M ., Fogelman, I. & Thomson, J. A. (1981) Clinical value of adrenal androgen measurement in the diagnosis of delayed puberty . Lancet, i, 689 -692. Cohen, H., Hay, I. D., Beastall, G. H. & Thomson, J. A. (1982) Failure of adrenal androgen to induce puberty in familial cytomegalic adrenocortical hypoplasia . Lancet, ii, 1471-1472. Delitala, G., Grossman, A. & Besser , G. M . (1981) Changes in pituitary hormone levels induced by Met-enkephalin in man - the role of dopamine. Life Sciences, 29, 1537-1544. De Wied, D. & Jolles, J. (1982) Neuropeptides derived from pro-opiocortin: behavioral, physiological, and neurochemical effects. Physiological Reviews, 62,976-1059. Dornhorst, A ., Jenkins, J . S., Lamberts, S. W. et al (1983) The evaluation of sodium valproate in the treatment of Nelson's syndrome. Journal of Clinical Endocrinology and Metabolism, 56.985-991. Ducharme, J . R. & Collu, R. (1982) Pubertal development: normal, precocious and delayed. Clinics in Endocrinology and Metabolism, 11 (I), 57-87 . Estivariz, F. E. , Hope, J., McLean, C. H . & Lowry, P. J. (1980) Purification and characterization of a y-melanotropin precursor from frozen human pituitary glands. Biochemical Journal, 191, 125-132 . Estivariz, F. E ., Iturriza, F., Mcl.ean, C . et al (1982) Stimulation of adrenal mitogenesis by N-lerminal proopiocortin peptides, Nature. 297,419-422. Farese, R. V. (1983) Phosphoinositide metabolism and hormone action . Endocrine Reviews, 4,78-95. Farese, R . V., Ling. N . C., Sabir, M. A. et al (1983) Comparison of effects of adrenocorticotropin and Lys-rj-melanocyre stimulating hormone on steroidogenesis, adenosine 3',5 'monophosphate production, and phospholipid metabolism in rat adrenal fasciculatareticularis cells in vitro. Endocrinology, 112. 129-132. Feek, C. M., Ratcliffe, J. G., Seth, J. et al (1981) Patterns of plasma cortisol and ACTH concentrations in patients with Addison's disease treated with conventional corticosteroid replacement. Clinical Endocrinology, 14,451 -458. Ferriss, J . B., Brown, J. J ., Fraser, R . et al (1981) Primary hyperaldosteronism. Clinics in Endocrinology and Metabolism, 10(3), 419-452. Fischer-Ferraro, C., Nahmod, V. E., Goldstein, D. J. & Finkielman, S. (1971) Angiotensin and renin in rat and dog brain. Journal ofExperimental Medicine. 133,353-361 . Fuller, R. W . (1981) Serotonergic stimulation of pituitary-adrenocortical function in rats. Neuroendocrinology, 32, 118-127. Ganguly, A ., Grim, C. E. & Weinberger, M. H. (1981) Anomalous postural aldosterone response in glucocorticoid-suppressible hyperaldosteronism . New England Journal of Medicine, lOS. 991-993.

616

C. M. FEEK, D. J . MARANTE AND C. R. W. EDWARDS

Ganten, D., Minnich, J. L., Granger, P . et al (1971) Angiotensin-forming enzyme in brain tissue. Science, 173,64-65. Grossman, A. & Clement-Jones, Y. (1983) Opiate receptors: enkephalins and endorphins. Clinics in Endocrinology and Metabolism, 12(1),31-56. Hayek , A., Crawford , J . D. & Bode, H. H. (1971) Single dose dexamethasone in the treatment of congenital adrenal hyperplasia . Metabolism, 20, 897-901. Himsworth, R. L., Lewis, J. G. & Rees, L. H. (1978) A possible ACTH secreting tumour of the pituitary developing in a conventionally treated case of Addison 's disease. Clinical Endocrinology, 9, 131-139. Hope, J., Ratter, S. 1., Estivariz, F. E. et al (1981) Development of a rad ioimmunoassay for an amino-terminal peptide of pro -opiocortin containing the y-MSH region: measurement and characterization in human plasma. Clinical Endocrinology, 15,221 -227. Horrocks, P . M. & London, D. R. (1982) Diagnostic value of 9 am plasma adrenocorticotrophic hormone concentrations in Cushing's disease. British Medical Journal, 285, 1302-1303. Hughes, I. A. (1982) Congenital and acquired disorders of the adrenal cortex. Clinics in Endocrinology and Metabolism, 11(1),89-125. lida, S., Itoh, Y., Moriwaki, K. et al (1981) Presence of ACTH-potentiating factors in rat anterior pituitary glands. Hormone Research, 14,155-164. James, V. H. T. (1979) Comprehensive Endocrinology: The Adrenal Gland. New York: Raven Press. 332 pp. Jones, M. T. (1978) Control of corticotrophin (ACTH) secretion. In The Endocrine Hypothalamus (Ed.) Jeffcoate, S. L. & Hutchinson, J. S. M. pp. 385-419. London, New York, San Francisco: Academic Press. Krieger, D. T. (1978) Factors influencing the circadian periodicity of ACTH and corticosteroids. Medical Clinics ofNorth America, 62(2),251-259. Lancranjan, I., Ohnhaus, E. & Girard, J. (1979) The alpha-adrenoreceptor control of adrenocorticotropin secretion in man . Journal of Clinical Endocrinology and Metabolism, 49, 227-230 . Laws, E. R ., Ebersold, M. J., Piepgras, D. G. et al (1982) The result s of transsphenoidal surgery in specific clinical entities. In Management of Pituitary Adenomas and Related Lesions with Emphasis on Transsphenoidal Microsurgery (Ed .) Laws, E. R., Randall, R. Y., Kern, E. B. & Abboud, C. F. pp. 277-305. Rochester: Mayo Clinic. Li, C. H. (1977) ACTH and related peptide hormones: chemistry, structure-function relationships; introductory remarks. Annals of the New York Academy of Sciences (USA), 297, 1-2. Ling, N. , Ying, S., Minick, S. & Guillemin, R. (1979) Synthesis and biological activity of four y-melanotropin peptides derived from the cryptic region of the adrenocorticotropin /fJIipotropin precursor. Life Sciences, 25, 1773-1779. Lis, M ., Hamet, P., Gutkowska, J . et al (1981) Effect of N-terminal portion of proopiomelanocortin on aldosterone release by human adrenal adenoma in vitro. Journal of Clinical Endocrinology and Metabolism, 52, 1053-1056. Lis, M., Lariviere, N., Maurice, G. et al (1982) Concomitant changes of ACTH, fJ-endorphin and N-terminal portion of pro-opiornelanocortin in rats. Life Sciences, 30, 1159-1164. Mains, R. E., Eipper, B. A. & Ling, N. (1977) Common precursor to corticotropins and endorphins. Proceedings of the National Academy of Sciences (USA), 74,3014-3018. Mangiapane, M. L. & Simpson, J. B. (1980) Subfornical organ: forebrain site of pressor and dipsogenic action of angiotensin II. American Journal of Physiology, 239, R382-R389. Matsuoka, H., Mulrow, P. J., Franco-Saenz, R. & Li, C. H . (1981) Effects of fJ-lipotropin and fJ-lipotropin-derived peptides on aldosterone production in the rat adrenal gland. Journal ofC/inicallnvestigation, 68,752-759. Mormede, P. (1983) The vasopressin receptor antagonist dPTyr(Me)AYP does not prevent stress-induced ACTH and corticosterone release . Nature, 302, 345-346. Nakanishi, S., Inove, A., Kita, T. et al (1979) Nucleotide sequence of cl'oned c DNA for bovine corticotrophin-fJ-lipotropin precursor. Nature, 278, 423-427. Nelson, D. H. (1980) ACTH, MSH and fJ-lipotropin. In The Adrenal Cortex: Physiological Function and Disease. pp. 24-47. Philadelphia, London, Toronto: W . B. Saunders.

HYPOTHALAMIC-PITUITARY -ADRENAL AXIS

617

New, M. I., Oberfield, S. E., Carey, R. et al (1982) A genetic defect in cortisol metabolism as the basis For the syndrome of apparent mineralocorticoid excess. Endocrinology of Hypertension: Serono Symposia, 50,85-102. Oki, A., Nakao, K., Tanaka, I. et al (1981) Concomitant secretion of adrenocorticotropin, f3-endorphin, and y-melanotropin from perfused pituitary tumor cells of Cushing's disease: effects of lysine vasopressin, rat median eminence extracts, thyrotropin-releasing hormone, and luteinizing hormone-releasing hormone. Journal of Clinical Endocrinology and Metabolism, 52,42-49. Oki, S., Nakao, K., Tanaka, I. et al (1982) Characterization of y-melanotropin-like immunoreactivity and its secretion in an adrenocorticotropin-producing mouse pituitary tumor cell line. Endocrinology, 111,418-424. Owerbach, D., Rutter, W. J., Roberts, J. L. et al (1981) The pro-opiocortin (adrenocorticotropin/f3-1ipotropin) gene is located on chromosome 2 in humans. Somatic Cell Genetics, 7,359-369. Parker, L. N. & Odell, W. D. (1979) Evidence For existence of cortical androgen-stimulating hormone. American Journal of Physiology, 236, E616-E620. Pedersen, R. C. & Brownie, A. C. (1980) Adrenocortical response to corticotropin is potentiated by part of the amino-terminal region of pro-corticotropin/endorphin. Proceedings ofthe National Academy of Sciences (USA), 77,2239-2243. Pedersen, R. C. & Brownie, A. C. (1983) Lys-vr-melanotropin binds with high affinity to the rat adrenal cortex. Endocrinology, 112, 1279-1287. Pedersen, R. C., Brownie, A. C. & Ling, N. (1980) Pro-adrenocorticotropin/endorphinderived peptides: coordinate action on adrenal steroidogenesis. Science, 308, 1044-1046. Pharn-Huu-Trung, M. T., de Smitter, N., Bogio, A. et al (1982) Responses of isolated guineapig adrenal cells to ACTH and pro-opiocortin-derived peptides. Endocrinology, 110, 1819-1821. Phillips, M . I. (1979) Biological effects of angiotensin in the brain. In Enzymatic Release of Vasoactive Peptides (Ed.) Gross, F. & Vogel, H. G. pp. 335-363. New York: Raven Press . Rees, L. H. (1977) Human adrenocorticotropin and lipotropin (MSH) in health and disease. In Clinical Neuroendocrinology (Ed.) Martini, L. & Besser, G. M. pp. 401-441. New York: Academic Press. Rees, L. H. & Lowry, P. J. (1979) Adrenocorticotrophin and lipotrophin. In Hormones in Blood (Ed.) Gray, C. H. & James, V. H. T. pp. 130-178. London, New York, San Francisco: Academic Press. Rivier, c., Rivier , J. & Vale, W. (1982) Inhibition of adrenocorticotropic hormone secretion in the rat by immunoneutralization of corticotropin-releasing factor. Science, 218, 377-379. Roberts, J . L. & Herbert, E. (1977) Characterization of a common precursor to corticotropin and f3-1ipotropin : cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule. Proceedings of the National Academy of Sciences (USA), 74, 4826-4830. Rosman, P. M., Farag, A., Benn, R. et al (1982) Modulation of pituitary-adrenocortical function: decreased secretory episodes and blunted circadian rhythmicity in patients with alcoholic liver disease. Journal of Clinical Endocrinology and Metabolism, 55,709-717. Seidah, N. G. & Chretien, M. (1981) Complete amino acid sequence of a human pituitary glycopeptide: an important maturation product of pro-opiornelanocortin. Proceedings of The National Academy ofSciences (USA), 78,4236-4240. Seidah, N. G. , Rochernont, J ., Hamelin, J. et al (1981) The missing Fragment of the prosequence of human pro-opiomelanocortin: sequence and evidence for C-terminal amidation. Biochemical and Biophysical Research Communications, 102,710-716. Shanker, G . & Sharma, R. K. (1979) f3-Endorphin stimulates corticosterone synthesis in isolated rat adrenal cells. Biochemical and Biophysical Research Communications, 86, 1-5. Shibasaki, T. & Masui, H. (1982) Effects of various neuropeptides on the secretion of proopiornelanocortin-derived peptides by a cultured pituitary adenoma causing Nelson's syndrome. Journal of Clinical Endocrinology and Metabolism, 55, 872-876. Shibasaki, T., Masui, H., Sato, G. et al (1981) Secretion pattern of pro-opiornelanocortinderived peptides by a pituitary adenoma from a patient with Cushing's disease. Journal of Clinical Endocrinology and Metabolism, 52,350-353.

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C. M. FEEK, D. J. MARANTE AND C. R. W. EDWARDS

Silas, L., Linton, E. A. & Lowry, P. J. (1983) Protein hormone and recombinant DNA unit. Abstracts of the 2nd Joint Meeting of British Endocrine Societies. University of York, 1983. p. 50. Smith, R., Donald, R. A., Espiner, E. A. et al (1980) The effect of different treatment regimens on hormonal profiles in congenital adrenal hyperplasia. Journal of Clinical Endocrinology and Metabolism, 51,230-236. Tanaka, I., Nakai, Y., Nakao, K. et. al (1981) I-melanotropin-like immunoreactivities in human pituitaries, ACTH-producing pituitary adenomas, and ectopic ACTH-producing tumours: evidence for an abnormality in glycosylation in ectopic ACTH-producing tumours. Clinical Endocrinology, 15,353-361. Thistlethwaite, D., Darling, J. A. B., Fraser, R. et al (1975) Familial glucocorticoid deficiency: studies of diagnosis and pathogenesis. Archives ofDiseases in Childhood, 50,291-297. Turkelson, C. M., Thomas, C. R., Arimura, A. et al (1982) In vitro potentiation of the activity of synthetic ovine corticotropin-releasing factor by arginine vasopressin. Pep tides, 1, 111-113. Vale, W, Spiess, J., Rivier, C. & Rivier, J. (1981) Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and {J-endorphin. Science, 213,1394-1397. Weiner, R. I. & Ganong, W. F. (1978) Role of brain monoamines and histamine in regulation of anterior pituitary secretion. Physiological Reviews, 58,905-976. Yamakado, M., Franco-Saenz, R. & Mulrow, P. J. (1982) A role of {J-melanotropin in the regulation of aldosterone secretion during sodium deficiency in the rat. Clinical Science, 63,93S-95S.