ACTH Synthesis Research Articles

Cushing's syndrome secondary to ACTH-secreting Wilms'Tumor

In a review of the literature we only found one case of Wilms'tumor associated with “ectopic ACTH syndrome”. We are reporting the second documented case. A girl, 7 1/2 years old, who 5 months ago presented a sudden gain in apetite and corporal weight. On admission she had the classical cushingoid appearance and a blood pressure of 160/120 mmHg. A large hard mass was palpable in the right side of the abdomen. The urography and renal arteriography demonstrated the presence of a right renal mass. Hormonal assays were consistent with Cushing'syndrome; the serum ACTH levels were extremely high. After surgical removal of the mass, we suspected it to be a stage I Wilms'tumor; this was confirmed through histopathological studies. Chemotherapy was initiated following the protocol of The National Wilms'Tumor Study Group. The girl quickly lost her cushingoid appearance and weight excess. Postoperative serum ACTH levels were normal. In order to explain the ectopic ACTH synthesis mechanism several theories are actually on discussion, such us loss of genetic depressor mechanisms, presence of abnormal DNA, and APUD system.

Some aspects of pituitary function after neuroadenolysis in patients with metastatic cancer.

The effects of neuroadenolysis on plasma titres of beta-endorphin, beta-lipotropin, ACTH, TSH and prolactin have been investigated in five patients with metastatic cancer who responded to the treatment and have been in remission for more than four years and in five others who were undergoing the treatment for the first time for pain due to cancer metastases. beta-Endorphin, beta-lipotropin and ACTH titres were within the normal ranges of values in both categories of patients but post-neuroadenolysis titres of these peptides were higher than those before the treatment. The ability to secrete TSH and prolactin and to respond to thyroid stimulating hormone releasing hormone (TRH) remains intact following the treatment. However, whereas basal TSH titres and response to TRH was lower in the majority of patients, no such effect was observed on prolactin secretion. Plasma titres of prolactin and TSH were below the sensitivity of the method in the five patients who are in remission for more than four years. These preliminary findings suggest that neuroadenolysis probably affects some mechanism(s) associated with the control of beta-endorphin, beta-lipotropin and ACTH synthesis.

Interaction between erythrocytes during aggregation studied by the scanning electron microscope

The levels of immunoreactive ACTH were determined in 109 pituitaries and in serum of human fetuses from the 7th to the 34th week of intrauterine life, and in serum of 7 infants. ACTH in the pituitary is detectable for the first time in a 7-week-old embryo. The mean content and concentration of ACTH in the pituitary significantly increases twice: between weeks 7-13 and between weeks 17-34, with a peak at the 34th week. Immunoreactive ACTH is detectable in serum from the 12th week of gestation. A significant rise was recorded at the 24th week, with maximum being reached by the 34th week. Serum ACTH decreases more than 10-fold by the time of the term as compared with that by the 34th week. The data obtained suggest that the pituitary of man starts ACTH synthesis from the 7th week and secrets it into fetal blood from the 12th week of prenatal development.

Adrenal corticosteroids and immunoreactive ACTH in chronically cannulated ovine foetuses with bilateral adrenalectomy.

Abstract. The adrenal fails to develop in the hypophysectomised ovine foetus but adrenal growth, maturation and increased cortisol secretion occur in the intact foetus whilst plasma immunoreactive ACTH remains at a constant value. The presence of a feedback relationship between foetal adrenal and pituitary was examined in 8 ovine foetuses which were bilaterally adrenalectomised (adrx.) at the time of implantation of catheters in the carotid artery and jugular vein (90–127 days of gestation). Ten chronically cannulated foetuses served as controls. Gestation length in these sheep was 147 ± 5 days. Plasma immunoreactive (IR) ACTH (IR.ACTH) was measured with a Sorin Biomedica kit. The antibody cross-reacts with ACTH1–39, and ACTH1–24, but not with ACTH1–13, ACTH18–39, β-LPH or β-endorphin. Cortisol (F) and corticosterone (B) were measured by a double isotope dilution derivative assay. In intact foetuses the IR.ACTH was 69.6 ± 7.1 pg/ml (mean ± sem, n = 37) until the day before parturition when values increased to 326 ± 60 pg/ml (n = 6). In adrx. foetuses of 91–121. 122–135 and > 136 days gestation the IR.ACTH values were 50.3 ± 4 (n = 14), 444 ± 46 (n = 26) and 1155 ± 175 (n = 6) pg/ml, respectively. The foetal F and B concentrations were 0.12 ± 0.027 μg/100 ml and 40 ± 6 ng/100 ml (n = 14), respectively, in adrx. foetuses. These values are not significantly different from those in intact foetuses younger than 135 days. The mean F and B concentrations in adrx. foetuses were 22 ± 4.3% (n = 14) and 49 ± 5.4% (n = 14) of the respective maternal values. In summary prior to 120 days the IR.ACTH, F and B concentrations are similar in intact and adrx. foetuses. After 122 days the IR.ACTH secretion by the foetal pituitary first begins to exhibit a feedback relationship with some adrenal secretion (?not F or B) and after 130–135 days some subtle change in pituitary sensitivity to F and/or B may operate. It is suggested that about 120 days a significant alteration in the foetal pituitary capacity for ACTH synthesis occurs.

A Longitudinal Hormonal Profile of the Genetically Obese Mouse*

Obese mice (C57BL/6J ob/ob) and their lean littermates were studied at various ages from immediately post weaning until 62 weeks of age, at which mortality increased markedly. Several age-related changes were noted. 1) Plasma glucose levels were elevated in obese mice 5-20 weeks and 62 weeks of age, but were similar to those in the lean mice at 20-60 weeks of age. Plasma insulin levels were elevated in obese mice, and there were no age-related differences. 2) Brain serotonin was elevated in obese mice at all ages and increased with age in both obese and lean animals. 3) Pituitary contents of ACTH and beta-endorphin were elevated in young obese mice and increased further as these mice approached their life expectancy. 4) The ratios of ACTH to beta-endorphin immunoreactivities were similar in obese and lean mice, except in obese mice over 50 weeks of age where this ratio was increased. We conclude that: 1) the obese mouse is characterized by hyperinsulinemia and hyperadrenocorticism throughout its life; 2) the insulin resistance of the obese mouse improves at 20 weeks of age, yet deteriorates as its life expectancy is approached; 3) the obese mouse has an elevated brain serotonin content similar to previously described elevations of the putative neurotransmitters dopamine and norepinephrine in these mice; and 4) as the obese mouse approaches its life expectancy, abnormalities may occur in the synthesis, processing, or secretion of ACTH and/or beta-endorphine.

Effect of cyproterone acetate on mouse adenohypophysis. I. The adrenocorticotrophs.

The adenohypophyses of adult male C57BL/6J mice pituitaries injected with cyproterone acetate for four weeks were examined with the electron microscope. The adrenocorticotrophs of the adenohypophysis showed an absence of the 200-nm granules that were lined along the plasma membrane, a decrease in profiles of rough endoplasmic reticulum, sparse distribution of ribosomes, and flatten Golgi membranes. It is believed the antiandrogen cyproterone acetate inhibited ACTH synthesis and release and that this was responsible for adrenocortical atrophy.

ACTH-Like Peptides, Pituitary-Adrenocortical Function and Avoidance Behavior

Publisher Summary ACTH and its analogues have appreciable affinity for stereo-specific opiate receptors in synaptosomal membranes of brain tissue. Interestingly, α-melanocyte stimulating hormone, vasopressin, substance P and thyroid releasing hormone proved to be inactive. These observations supported the view that ACTH-like peptides (ALP) interfere with morphine in the central nervous system. In addition to this, the findings are in accordance with observations that ALP can antagonize the analgesic effects of opiates. The discovery of behavioral effects of ALP had a profound influence upon the study of endocrine control over adaptive behavior, and greatly contributed to the development of research into neuropeptides and peptidergic transmission in the brain. Considerable evidence suggests that ALP may act upon brain protein synthesis and catecholamine turnover, and that they bind to opiate receptors. The experimental observations that ALP of extra-pituitary origin are present in the brain led to the assumption that such neuropeptides are involved in neural transmission. The biologically active and immunoreactive ACTH content of the brain remained unchanged after either hypophysectomy or dexamethasone administration. These last observations indicate that other factors are involved in regulation of pituitary ACTH synthesis than that of the brain ALP. Whether the neuropeptides are directly involved in neural transmission or rather are playing a modulatory role, is still the subject of investigations.

Immunocytochemical study of neurohypophysial peptides during corticotropic maturation of infant rats

Immunocytochemical localization of vasopressin (VP), oxytocin (OXY) and neurophysins I and II (NI and NII) in the hypothalamus of growing rats revealed several new features. Hormones and neurophysins were present in magnocellular neurons of newborn rats: VP and NII in a first neuronal population, OXY and NI in a second neuronal population. For the first three days after birth, the ratio of detected VP to detected NII increased whereas the ratio of detected OXY to detected NI appeared constant. It was obvious that maturation of the OXY/NI magnocellular system occurred later than maturation of the VP/NII magnocellular system. During the first three weeks of development, VP/NII fibres in the median eminence were separated into two distinct groups: the hypothalamo-neurohypophysial tract in the internal part of the median eminence, and pericapillary endings heavily loaded with VP and NII in the external part of median eminence. OXY/NI fibres did not show this particular distribution. Therefore, during this post-natal period, loading of infundibular VP endings was related to the increase of ACTH synthesis demonstrated by various authors. Therefore, we suppose that simultaneous participation of VP and corticotropin-releasing hormone does not only appear during experimental and chronic stimulation of ACTH synthesis (after adrenalectomy, for example) but that it also exists during physiological stimulation of corticotropic function.

Inhibition of the Pituitary-Adrenal Response to Stress during Deprivation-Induced Feeding

Plasma corticosterone and plasma and pituitary ACTH concentrations were determined during feeding and after application of an acute stress at various times after food and water presentation to male rats maintained on a restricted feeding and watering schedule. Both plasma corticosterone and ACTH concentrations fell after the presentation of food and water, and this fall was accompanied by increased levels of ACTH in the pituitary gland. In addition, a rise in plasma levels of ACTH was inhibited in response to an acute stress applied at 0--5 min after presentation of food and water, but ACTH synthesis was not. This inhibition of ACTH and corticosterone secretion in response to stress was transient and dissipated as a relatively linear function of the interval between food presentation and application of the stress. The results suggest that this feeding-induced, corticosteroid-independent inhibition of pituitary-adrenal activity involves active inhibitory mechanisms operating initially on ACTH secretory processes of the pituitary and later on the synthesis of ACTH or on the secretion of hypothalamic corticotropin-releasing factor.

Compared Localizations of Prolactin-Like and Adrenocorticotropin Immunoreactivities within the Brain of the Rat

By an immunoperoxidase (PAP) technique, ACTH- and prolactin-like substances were detected in neurons of several hypothalamic nuclei in young and adult rats. Neurons in the arcuate and ventromedial nuclei were simultaneously both ACTH-like and PRL-like positive. Labelled fibres were followed to their fields of projections. Intensity of labelling was not influenced by experimental procedures known to change PRL and ACTH content of the pituitary. Colchicine enhanced labelling of perikarya while lowering that of fibres. The results support neuronal synthesis of hormonal ACTH and PRL related molecules and their involvement in the control of autonomic nerve functions.

Effect of indomethacin and adrenocorticotrophic hormone on renal function in man: An experimental model of inappropriate antidiuresis

The effect of prostaglandin synthesis inhibition on basal and ACTH-stimulated adrenal and renal function was investigated in normal volunteers. Data were collected during control and experimental study periods (13 days each). Adrenocorticotrophic hormone (Cosyntropin, 80 U/day) was administered i.v. on days 8 and 9 of each period. Indomethacin (150 mg/day) was given on days 5 through 13 of the experimental period. The subjects ate a constant diet containing 9 mEq of sodium, 100 mEq of potassium, and 2,500 ml of fluid daily. Indomethacin markedly inhibited urinary PGE excretion and plasma PGE concentration. The effect of ACTH alone as compared to the effect of ACTH and indomethacin showed: plasma sodium concentration, 139 +/- 1 vs. 131 +/ 3 mEg/liter (P less than 0.01, mean +/- SEM); plasma osmolality, 287 +/- 3 vs. 270 +/- 3 mOsm/liter (P less than 0.01); free water clearance, 97 +/- 66 vs. -1100 +/- 380 ml/24hr (P less than 0.01); urine volume, 2,000 +/- 60 vs. 950 +/- 200 ml/day (P less than 0.01); and urine osmolality 282 +/- 12 vs. 720 +/- 144 mOsm/liter (P less than 0.01). We conclude that the effects of ACTH and prostaglandin synthesis inhibition interact to result in inappropriate antidiuresis.

Effects of Hypothalamic Extract and Corticosterone on Incorporation of <sup>14</sup>C-Phenylalanine into Newly Synthesized ACTH-Like Protein <i>in vitr</i><i>o</i>

The purpose of the present experiments was firstly, to examine the efficacy of the oxycellulose-Amberlite CG-50 extraction procedure for the isolation of rat pituitary ACTH and secondly, to study the effects of both hypothalamic extract and corticosterone on the synthesis of rat pituitary ACTH. Briefly, 'cold' rat pituitary ACTH was monitored by bioassay in each step of the isolation procedure as a function of corticosterone stimulation in incubated adrenal slices. Labeled ACTH was isolated from rat pituitaries previously incubated in vitro and obtained from adrenalectomized animals and cortisone acetate treated animals to provide further data to substantiate that the labeled isolated protein contained ACTH. Cold ACTH was detected in two purified fractions (oxycellulose and Amberlite CG-50). Adrenalectomy stimulated and cortisone acetate suppressed the incorporation of 14C-phenylalaine into the ACTH-like protein in these two fractions. A continuously increasing uptake of the label into ACTH-like protein in the isolated fractions occurred with time providing evidence that de novo synthesis had taken place. Corticosterone inhibited ACTH synthesis at both 10(-4) and 10(-6) M, whereas, hypothalamic stalk median eminence (HSME) caused a significant, but not dose-related, increase in ACTH synthesis with 3/5, 1, and 2 HSME equivalents added.

Solid Phase Peptide Synthesis by Oxidation-Reduction Condensation. Synthesis of Adrenocorticotropin(1–24) by Chain Elongation at the Carboxyl End on Solid Support

Abstract Solid phase peptide synthesis by oxidation-reduction condensation via fragment condensation at the carboxyl end was established by a new monitoring method based on high pressure liquid chromatography. The merit of the method was demonstrated through the synthesis of ACTH (1–24). A resin–tetracosapeptide with quantitative amino acid ratios was easily prepared by coupling 6 fragments of 1–2, 3–7, 8–10, 11–14, 15–19, and 20–24 sequences. ACTH (1–24) with a full activity was obtained in 25% yield from the initial resin-dipeptide by hydrogen fluoride treatment and purification with carboxymethyl-cellulose chromatography.

Corticotroph cells in primary cultures of rat adenohypophysis: A light and electron microscopic immunocytochemical study

Monolayer cultures of trypsin-dispersed cells of the rat adenohypophysis were grown for 5 to 54 days. ACTH was localized by immunocytochemistry using an antiserum to synthetic ACTH1-28 prepared in rabbit and sheep anti-goat immunoglobulin coupled with peroxidase. ACTH content of the culture medium was measured by radioimmunoassay. Corttion time. The corticotrophs retained their essential morphological characteristics. Immunological staining was found in the secretory granules, some tubular or saccular structures, parts of the rough endoplasmic reticulum, and the cytoplasmic matrix. Immature secretory granules in the Golgi apparatus as well as some Golgi elements showed different degrees of immunoreactivity. In agreement with the high ACTH content of the culture medium the number, size and shape of the secretory granules, the active Golgi apparatus, the high amount of extragranular ACTH as well as pictures suggesting granule extrusion claim for a high ACTH synthesis and transport (and low ACTH storage) in the cultured corticotrophs.

INTERRELATIONSHIPS OF PITUITARY AND PLASMA CORTICOTROPHIN AND PLASMA CORTICOSTERONE DURING ADRENOCORTICAL REGENERATION IN THE RAT

Pituitary and plasma ACTH and plasma corticosterone were measured at various times after adrenal enucleation or adrenalectomy. The changes in ACTH could be inversely correlated with those in plasma corticosterone. They could also be prevented by maintenance therapy with the steroid. The results indicate that the corticosteroids control both the release and the synthesis of ACTH but that their effect is predominantly on the release of the hormone.

ACTH content of pituitary glands of arteriosclerotic breeder vs. non-arteriosclerotic virgin rats

Acid extracts were made of the pituitary glands of non-arteriosclerotic male and female virgin rats and arteriosclerotic, male and female breeder rats. The ACTH content of these pituitary glands was determined by measuring the amount of corticosterone produced by the adrenal glands of healthy but hypophysectomized rats when challenged by the various ACTH acid extracts. The pituitary glands of the arteriosclerotic animals were significantly heavier than non-arteriosclerotic rats and contained decreased amounts of ACTH commensurate with the severity of the pituitary donor's arteriosclerosis. Similarly, arteriosclerotic donors had heavier adrenal glands but contained considerably less corticosterone than non-arteriosclerotic virgin rats. Male breeder rats developed microscopic aortic lesions involving intimal mesenchymal cell and ground substance alterations whereas the female breeder lesions progressed from intimal ground substance and connective tissue changes to medial elastolytic degeneration and eventual medial cartilaginous and osseous metaplasia. Histopathologic examination of the pituitary and adrenal glands demonstrated hyperplasia of basophil and other cytologic elements of the pituitary gland including colloid-filled cysts and lipid depletion of the zona glomerulosa as well as hemorrhage and thrombosis of the adrenal cortex of the arteriosclerotic breeder rats. The hypothesis is made that repeated breeding leads to abnormal hypothalamic regulation of pituitary gland synthesis and release of ACTH, which, in turn, contributes to abnormal adrenal glandular function conditioning the aortic wall toward abnormal changes in mesenchymal cell activity and metabolism of connective tissue ground substance.

In VitroRelease and Biosynthesis of Tumor ACTH in Ectopic ACTH Producing Tumors

Tumor tissues obtained from 4 patients with the ectopic ACTH syndrome were studied for release and synthesis of tumor ACTH, using an in vitro incubation system. The effect of various agents on release of tumor ACTH was evaluated in three cases; beta-MSH released and adenosine 3',5'-monophosphate (cyclic AMP) formed in the tissue were determined in one. Biosynthetic experiments using labeled amino acid incorporation were performed in two cases. Secretion of tumor ACTH was significantly stimulated in all cases by crude rat median eminence extract which was also effective in stimulating beta-MSH secretion associated with elevated tissue cyclic AMP levels in one. Addition of cyclic AMP and dibutyryl cyclic AMP caused a significant increase in release of both tumor ACTH and beta-MSH in one. Biogenic amines (norepinephrine and serotonin) markedly elevated tussie cyclic AMP levels without a corresponding increase of hormone release in one. Incorporation experiments revealed that 3H- or 14C-phenylalanine was incorporated into immunoreactive ACTH of a larger molecular size (big ACTH) in both cases by chromatographic procedures. However, biological activity of big ACTH was found to be undetectable by an in vivo steroidogenic assay. A mild tryptic digestion of the big forms resulted in the appearance of little ACTH to which the major radioactive peak shifted. These data suggest that the mechanism of release of tumor ACTH and beta-MSH is very similar to that of the pituitary, and that intracellular cyclic AMP may in part play some role in release of both hormones. It is also suggested that some ectopic ACTH producing tumors predominantly synthesize big ACTH, a possible precursor of ACTH, with less bioactivity.

Pituitary Nuclear Retention of Dexamethasone and ACTH Biosynthesis

The possible binding of ³H-dexamethasone (Dx)to the anterior pituitary and the effect of the steroid on neo-synthesis and release of ACTH of glands incubated with crude CRF extracts were examined. Neosynthesis of ACTH was determined by immunoprecipitation after incorporation of ¹⁴C-phenylalanine into the peptide. Using a cell-free system, ³H-Dx was shown to combine with the nuclear fraction of the pituitary. Short-term <i>in vitro </i>pre-treatment with Dx suppressed the CRF-induced hormonal release, but did not alter ACTH biosynthesis. The releasing mechanism, on the other hand, did not appear to be related to <i>de novo </i>synthesis. Prolonged <i>in vivo</i> administration of high concentrations of Dx initially caused a decrease of ACTH synthesis and, later, also impaired the synthesis of total proteins. Synthesis and release of ACTH may be dissociated, but there is a possibility that a correlation exists between these two processes. On the other hand and as far as the direct effect of Dx on the pituitary is concerned, the nuclear uptake of the steroid probably reflects an early step in the modulation of corticotropic activity.

CRF-stimulated biosynthesis of ACTH in a cell-free system from rat anterior pituitaries

A cell-free system consisting of ribosomes, pH 5 enzymes and supernatant prepared from rat anterior pituitaries was found to be active in the incorporation of 3H-serine into ACTH. The rate of biosyntesis of ACTH, in a cell-free system as, measured by the incorporation of radioactive amino acid, and the rate of biological activity were markedly increased by the addition of CRF. The synthesis of ACTH was significantly inhibited by puromycin and RNAase but was not significantly inhibited by actinomycin D and DNAase.

Adrenal and plasma corticosterone and pituitary and plasma ACTH in alloxan diabetic rats.

SummaryPlasma and adrenal corticosterone as well as ACTH in both plasma and pituitaries of acutely diabetic rats were measured 72 hr after intraperitoneal injection of alloxan (20 mg/100 g). ACTH assays were performed in dexamethasone suppressed rats. A 50% increase in adrenal weights was associated with a 90% increase in total adrenal corticosterone and a 67% increase in adrenal corticosterone concentration. The high level of adrenal activity was associated with an equally high level of plasma corticosterone as well as plasma ACTH. The pituitary ACTH reserve remained unchanged. Experimental diabetes is associated with increased pituitary–adrenal activity without the depletion of the pituitary ACTH reserve. In the basal state of uncontrolled diabetes, ACTH synthesis is capable of keeping pace with the rate of ACTH release.