ACTH Synthesis Research Articles

Establishment of human malignant melanoma clonal cell lines that secrete ectopic adrenocorticotropin.

THE study of factors regulating the secretion of adrenocorticotropin (ACTH) by non-pituitary tumours in patients with the “ectopic ACTH syndrome”1 is complicated by many factors, not the least of which is the presence of the normal pituitary gland, which also secretes ACTH. Furthermore, although ectopic ACTH resembles pituitary ACTH in a variety of test systems2, proof of identity requires complete chemical characterization and only rarely are tumours found to contain as much as 1 mg of ectopic ACTH3. For these reasons, availability of continuous clonal tumour cell lines that maintain their functional capacity for biosynthesis of ectopic ACTH in tissue culture would represent a major advantage for studying the factors regulating the synthesis and secretion of ectopic ACTH, as well as determining the precise structure of the ectopic hormone.

Electron microscopic-immunocytochemical localization of adrenocorticotropin and melanocyte stimulating hormone in the pars intermedia cells of rats and mice

Electron microscopic localization of adrenocorticotropin (ACTH) and melanocyte stimulating hormone (MSH) in light, dark and ACTH cells in the pars intermedia (PI) of rats and mice is attempted by using antisera to βp 1–24, βp 17–39 ACTH and βb MSH with the immunoglobulin-peroxidase bridge technique. All of the PI parenchymatous cells (light, dark and ACTH cells), except the marginal cuboidal and the ependymal like cells, in rats and mice show very good localization of ACTH and MSH staining. In the light and dark cells, stain of varying intensity is seen on the secretory granules, vesicles and also in many places on the surface of the rough endoplasmic reticulum. There is no staining on the mitochondria, in the nuclei or in the granules inside and around the cisternae of the Golgi complex. Dark stained dense core granules become larger and larger as they appear farther and farther away from the Golgi complex. On the other hand, in the ACTH cells of the PI, ACTH antisera show stronger stained granules in the Golgi complex including the cisternae, similar to the pars distalis (PD) ACTH cells. From these observations it is concluded that the corticotropin in light and dark cells, is not packaged or condensed in the Golgi complex like that in the ACTH cells. MSH synthesis in light and dark cells also seems to be similar to that of ACTH synthesis. It is likely that the granules accumulate ACTH and MSH secretions after they are liberated from the Golgi cisternae, and thus become bigger and bigger in size. In case of ACTH cells of PI and PD, corticotropin may be packaged in Golgi cisternae and the size of the granule does not change much. This shows that there are distinct immunocytochemical differences between the light, dark and ACTH cells of the PI. At the moment, it is difficult to say whether ACTH and MSH are present in the same granule or not.

Effects of the pineal gland in unilaterally adrenalectomized rats.

ABSTRACT The following experiments were undertaken to determine the effects that the pineal secretory substance might have on compensatory adrenal hypertrophy and corticosterone plasma levels in unilaterally adrenalectomized rats. It was demonstrated that blinded animals and animals administered with a bovine pineal extract exhibited a reduction in the percentage compensatory adrenal hypertrophy, a reduction in the thickness of the zona fasciculata, and a reduction in the plasma corticosterone level. These inhibitory effects were reversed by pinealectomy and ACTH injections, suggesting that the pineal gland, acting as a mediator for environmental lighting, might influence synthesis and/or release of ACTH.

Chlorpromazine: Inhibition of ACTH and cyclic 3′,5′-AMP stimulated corticosterone synthesis

Chlorpromazine inhibits the ACTH and cyclic AMP-stimulated corticosterone synthesis, invitro, in the quartered rat adrenal glands without greatly affecting the basal production of corticosterone. Tissues exposed for 30 minutes to cyclic AMP do not show inhibition of cyclic AMP stimulation by chlorpromazine and the removal of chlorpromazine from the incubation medium restores the tissue response to cyclic AMP.

Effect of adrenalectomy and cortisol on the in vivo rate of incorporation of phenylalanine-14C into rat corticotropin.

A method has been developed for studying the rate of in vivo ACTH synthesis by measuring the incorporation of phenylalanine-¹⁴C into ACTH in the rat. At 1 h after the intra-peritoneal injection of labeled amino acid the anterior pituitary was obtained and ACTH was extracted with hot glacial acetic acid, precipitated with ether, and adsorbed onto oxidized cellulose. The ACTH fraction was then eluted with 0.1 N hydrochloric acid. This fraction was shown to contain radioactive ACTH and did not contain free phenylalanine-¹⁴C. Changes in the rate of ACTH synthesis were estimated by changes in specific activity of the ACTH following a 1 h exposure of the rat to isotope. At 7, 14, and 24 days following adrenalectomy the rate of pituitary ACTH labeling doubled, while at 40 days, there was a three- to fivefold increase over the control rate. Administration of cortisol to the adrenalectomized but not to the intact rat decreased the rate of pituitary ACTH labeling. Nearly total inhibition of ACTH synthesis was caused by cortisol (0.5 mg/100 g body wt.) within 2 h. It was found that changes in the rate of incorporation of labeled amino acids into ACTH were not a reflection of general changes in the rate of tissue protein synthesis.

PITUITARY-ADRENAL FUNCTION IN TEN PATIENTS RECEIVING MEDROXYPROGESTERONE ACETATE FOR TRUE PRECOCIOUS PUBERTY

ABSTRACT The effect of medroxyprogesterone acetate (MPA) on pituitary-adrenal function was investigated in 10 patients with true precocious puberty. Although the patients receiving MPA responded to exogenously administered ACTH by a rise in the plasma 17-hydroxycorticosteroids, the mean values of both the pre- and post-ACTH levels were statistically lower than those obtained in the controls. In 7 of the 8 patients the increase of the urinary 17-ketogenic steroids following the oral administration of metyrapone was less than 2-fold. The data suggest that MPA in the dosages tested has a suppressive effect on pituitary ACTH synthesis and/or release. Despite this inhibition, however, no clinical evidence of hypoadrenocorticism was observed in these patients even during periods of stress. When the drug was discontinued, pituitary-adrenal function rapidly returned toward normal, suggesting that the suppressive effect of MPA was a relatively mild one.

ACTH synthesis and release in pituitary monolayer culture: effect of dexamethasone.

ACTH synthesis and release in pituitary monolayer culture: effect of dexamethasone.

ACTH and protein synthesis in a transplantable ACTH-secreting tumor. Evidence for the absence of MSH.

The synthesis of ACTH and protein was studied in vitro in a transplantable mammotropic pituitary tumor (MtT.F4) in female Fisher rats. In tumor-bearing rats the thymus was involuted and the adrenals were 15 times normal weight. Based on radioimmunoassay, the ACTH concentration in MtT.F4 was at least 10-fold less than the concentration of the hormone in the normal pituitary. Crude ACTH was isolated from MtT.F4 by extraction with HC1, heat coagulation, and oxycellulose adsorption. The hormone was purified by gel filtration and carboxymethylcellulose chromatography. In vitro incubations were performed with MtT.F4 and with normal bovine anterior pituitary. The incorporation of radioactive phenylalanine into both ACTH and protein was about 3-fold greater in the tumor than in the normal pituitary. However, the relative rate of amino acid incorporation into ACTH, compared to that into protein, was no greater in the tumor than in the normal pituitary. These data suggested that ACTH was secreted 30–300 times more rapidly from the tumor than from the normal pituitary. When extracts from MtT.F4 were assayed for MSH content by the frog-skin assay, all the activity could be accounted for by the intrinsic melanocytestimulating activity of the ACTH present in the tumor. MtT.F4 was incubated with radioactive phenylalanine, and extracts were analyzed chromatographically for MSH. No evidence was found of radioactivity or optical density where either α- or β-MSH is normally found. It was concluded that MtT.F4 in female Fisher rats produces ACTH but not α- or β-MSH. (Endocrinology 86: 1394, 1970)

Pituitary-adrenal relationships in mice with hereditary nephrogenic diabetes insipidus, with special emphasis on the neurohypophysis and pars intermedia.

The extremely hypertrophic and hyperplastic pars intermedia (PI), of the hereditary nephrogenic diabetes insipidus (DI Os/+) mice, which is correlated to the increased neurosecretory activity of the hypothalamo-hypophysial system on one hand, and the hypertrophy of the adrenal cortex on the other, is compared to these structures in normal VII+/+ mice. In DI mouse, in addition to the moderate hypertrophy of the pituitary gland as a whole, other unique features include a great penetration of neurohypophysial fibres in PI and the presence of a great number of light glandular cells. Very often, these glandular cells are surrounded by neurosecretory fibres, pituicyte-like fibres or AF+ granules. The small marginal cuboidal cells bordering the hypophysial cleft contain and/or are surrounded by AF+ granules. These features are lacking in normal VII+/+ mice. The excess release of NSM from the neurohypophysis by different stresses such as dehydration, formalin injection, surgery and adrenalectomy is accompanied by a hypertrophy of PI and of the light glandular cells as well as of the adrenal cortex in the normal and DI mice. In DI mice under different conditions of stress, all the AF+ granules in different parts of PI disappear in addition to the above changes. From the present experimental evidence and from the fact that DI mice have a hypertrophied hypothalamus (neurohypophysis), pituitary and adrenal cortex, it appears that these three endocrine entities are functionally related. The role played by vasopressin in augmenting MSH, CRF and ACTH is discussed. The probable role of light glandular cells of PI in synthesis of ACTH or ACTH-like substances is also discussed.

In vivo studies on the mechanism of action of 6-dehydro-16-methylene-hydrocortisone (STC 407) on the hypothalamo-pituitary-adrenal axis in rats.

The mechanism of action of a new synthetic steroid, 6-dehydro-16-methylene-hydrocortisone (STC 407), which has been found useful in the treatment of Cushing’s syndrome due to adrenal hyperplasia, was studied in the rat. After treatment of normal rats with STC 407 for 11–14 days, the CRF content of the median eminence of the hypothalamus (SME), the concentration of ACTH in the anterior pituitary, the wet weight of the adrenal glands/ 100 g b.w., the adrenal corticosterone concentration/100 mg, and the plasma corticosterone level were significantly decreased. After treatment of adrenalectomized rats with STC 407 for 4 days, the concentration of ACTH in the pituitary was increased, but there was no appreciable change in CRF activity in the SME. Plasma levels of ACTH, which were elevated after adrenalectomy, decreased to undetectable levels after 4 days’ treatment with 400 <i>µ</i>g/100 g b.w. daily. Treatment of adrenalectomized rats with this steroid for 13 days considerably reduced CRF activity in the SME, and significantly raised the pituitary ACTH content. These results indicate that STC 407 suppresses both the release and the synthesis of ACTH by the pituitary in normal rats, this effect being mediated at least in part by a reduction in hypothalamic CRF activity. In adrenalectomized rats, the predominant effect of this steroid seems to be suppression of release of ACTH by the pituitary.

ACTH synthesis and release in septal-lesioned rats exposed to air shuttle-avoidance

Male Sprague-Dawley rats were either sham-operated or bilaterally lesioned in the lateral septal area. One half of each group was trained to a maximum of 3 days in a shuttlebox utilizing pressurized air US. Only 1/7 sham-operated rats met the learning criterion (5 avoidance responses in 6 trials) while 4/7 septal-lesioned rats did so. There was a significant increase in the percentage avoidance response and the mean avoidance latency of the lesioned animals, but no change in the mean escape latency. ACTH content was increased in rats with bilateral septal lesions while ACTH release, as measured by both plasma corticosterone concentration and by adrenal weight, was increased after exposure to air shuttle-avoidance.

Increased ACTH release without increased synthesis.

Male white rats were treated with saline (controls) or puromycin or dexamethasone and morphine and subjected to laparotomy. Corticosterone secretion rates were measured at intervals for 30 to 120 min following incision. Incorporation of 14C-valine into pituitary and adrenal protein and polypeptides served as an index of the effect of experimental agents on ACTH synthesis. In control animals corticosterone secretion averaged 0.6-0.7 μg/min following laparotomy and incorporation of 14C-valine increased roughly linearly with time. Corticosterone secretions (0.2-0.3 μg/min) of dexamethasone- morphine blocked rats were inhibited, but incorporation of 14C-valine was similar to that of control rats. In the puromycin-treated group, both corticosterone secretion (0.2 μg/min) and incorporation of labeled valine were inhibited, but plasma ACTH levels (3.8 mU/100ml) and plasma ACTH half-time (3.0 min) were similar to those of the control animals. Pituitary ACTH content of puromycin-treated animals 2 hr after laparoto...

Demonstration of alpha-glycerophosphate dehydrogenase in the human pituitary gland with special reference to the cells of the pars tuberalis.

The demonstration of α-glycerophosphate dehydrogenase (α-GPD) in epithelial cells of the pars tuberalis in man is reported. This method provides a clear means of staining these usually poorly displayed cells. The proximal, or cranial, terminus of these cells appears to be at the median eminence. Caudally, within the pituitary gland proper, the cells seem to blend with the basophilic (periodic acid-Schiff positive) cells of the pars distalis, many of which are also α-GPD positive. Staining is limited to the cytoplasm of the positive cells; there is no nuclear staining and there is no staining of connective tissue, colloid and other epithelial cells. The widespread distribution of the α-GPD positive cells in the human pituitary weakens our earlier hypothesis, based on results in the rat, that this enzyme might be a histochemical hallmark of TSH production or release. Its limitation to periodic acid-Schiff positive cells suggests its association with the cellular sites of TSH, FSH, LH or ACTH synthesis. (End...

A "short" feedback loop in the control of ACTH secretion.

Permanent ACTH implants (USP Reference Standard and synthetic β1-24- corticotrophin) into the median eminence significantly depress blood corticosterone levels in the rat, suggesting that a “short” feedback loop might link synthesis and/or release of ACTH to pre-existing levels of the hormone. (Endocrinology 77: 392, 1965)

EFFECT OF HYPOXIA ON SECRETION OF ACTH IN THE RAT.

Body weights, organ weights, and blood and pituitary ACTH concentrations were determined in rats exposed continuously to 20.9, 15.0, or 10.0% O2 atmospheres at normal barometric pressure for periods ranging from 3 to 120 hr. Hypoxia resulted in some loss of body weight, and loss of weight of the thymus and the ovary. The weight of the adrenals increased. Blood ACTH rose rapidly after exposure to hypoxic conditions, remaining elevated for the duration of the experiment. Control animals (20.9% O2) placed in the same chamber had detectable blood ACTH only at the 3-hr sampling time. Pituitary ACTH also rose rapidly during hypoxia, the elevation being greatest with the most severe hypoxic state. The rate of ACTH synthesis and secretion in the resting state and during the stress of hypoxia is discussed.

EFFECT OF RAT MEDIAN EMINENCE EXTRACTS ON PITUITARY ACTH CONTENT IN NORMAL AND ADRENALECTOMIZED RATS.

The pattern of the changes in the concentration of adenohypophysial ACTH after a single intracarotid injection of median eminence extract was studied in normal and adrenalectomized rats. In normal animals pituitary ACTH content increased slightly 1.25 min after the injection, but subsequently decreased, reaching a concentration 36 % that of control animals 40 min later. Increasing the dose of median eminence extract caused a very marked increase in pituitary ACTH concentration 1.25 min after its administration. In adrenalectomized rats the concentration of ACTH in the pituitary showed a 3- fold increase within 2.5 min and had returned to the pre-injection level within 40 min of the administration of the extract. It is concluded that the close similarity in the pattern of the changes in pituitary ACTH content after stress or after the administration of median eminence extracts suggests that the median eminence plays an important role in bringing about the changes in ACTH synthesis and secretion caused by s...

Effect of Acute Stress on the Pituitary Gland: Action of Ethionine on Stress-Induced ACTH Release1

The effect of subcutaneous administration of ethionine was studied on stress-induced ACTH release in female rats. Ethionine was shown not to affect adrenal ascorbic acid content, pituitary ACTH content, or the adrenal ascorbic acid depleting effect of test doses of ACTH. Ethionine produced a marked suppression of ACTH release following ether-laparotomy stress. The suppression was most marked 1 hr after ethionine injection. The effect of ethionine was antagonized by simultaneous administration of methionine. Ethionine also suppressed stressinduced ACTH release in 1 day and 30 day adrenalectomized rats, but did not affect the resting level of blood ACTH. Ethionine prevented the stress-induced rise in pituitary ACTH content of adrenalectomized rats, but did not affect the resting level of ACTH in these rats. It was concluded that ACTH release after stress involved the new synthesis of ACTH, while the basal or resting secretion of ACTH was a function of the pituitary stores of this hormone.

Effect of acute stress on the pituitrary gland: uptake of serine-1-C14 into ACTH.

The effect of acute stress on the incorporation of serine-1-C14 into the anterior and posterior pituitary lobes and the hypothalamus was studied. Stress produced an increased uptake of the labeled amino acid into the glacial acetic acid extract of the adenohypophysis. Using 2-dimensional electrophoretic and chromatographic separation on paper sheets, radioactive extracts were characterized. Every extract demonstrated the presence of the same radioactive peptide (Spot 1). The amount of radioactive incorporation into this spot was increased by stress. Using biological assay methods, Spot 1 was identified as ACTH. The electrophoretic and chromatographic characteristics of rat ACTH were markedly different from those of porcine or ovine ACTH. It was concluded that the effect of acute stress was to cause an increased synthesis of ACTH in the adenohypophysis of rats.

Effect of CRF on maintenance of the adrenocorticotrophic function of anterior pituitary grafts.

SUMMARY Rats were hypophysectomized and their own anterior pituitary glands transplanted under the kidney capsule. Effects of chronic administration of corticotrophin-releasing factor (CRF), lysin-vasopressin and α-melanocyte stimulating hormone (α-MSH) on the adrenocorticotrophic activity of the pituitary grafts were studied using as criteria the concentration of ACTH in the grafts, resting levels of circulating plasma corticosterone, adrenal weight and also response of the graft to a challenge dose of CRF measuring plasma and adrenal corticosterone levels 12 min. later. The results show that prolonged administration of CRF has maintained synthesis of ACTH by the transplanted pituitary as well as its ability to release ACTH when acutely stimulated by CRF. These results were not duplicated by the administration of lysin-vasopressin or α-MSH in conditions identical to the ones used for studying CRF, and using doses equivalent to the vasopressor and melanophoretic activities of this particular preparation of CRF.

Correlation of Pituitary Ribonucleic Acid Levels and ACTH Production Following Adrenalectomy and Cortisone Administration.

SummaryThe concentration of RNA in pituitary tissue was determined following various periods of adrenalectomy and prolonged cortisone treatment. Elevations of pituitary RNA at 1/2 and 48 hours post-operatively correlate with increased tissue content of ACTH reported for the post-adrenalectomy periods. Reduction in level of tissue RNA following cortisone administration likewise correlates with decreased amounts of ACTH in pituitaries reported after such treatment. The data, utilizing tissue RNA as an index of protein hormone synthetic activity. provide further information that indicate adrenal cortical steroids regulate synthesis of ACTH.