Molecular Weight Forms Of ACTH Research Articles

ACTH precursors: biological significance and clinical relevance.

ACTH is synthesized as part of the precursor proopiomelanocortin or POMC (Fig. 1). In the human pituitary POMC is enzymatically processed to pro-ACTH, which is then cleaved to give ACTH. Processing of POMC in the pituitary takes place within the regulated secretory pathway and is accomplished by the protease PC1 located within mature dense core granules (Benjannet et al., 1991; Smeekens et al., 1991). The presence of ACTH precursors in plasma was first demonstrated in normal subjects after stimulation with metyrapone (Yalow & Berson, 1971) and was later observed after insulin-induced hypoglycaemia (Hale et al., 1986). However, complex chromatographic techniques were required to separate ACTH precursors from ACTH. Clearly, this approach could not be used for large numbers of patient samples and would not provide a quantitative assessment of the concentrations of the ACTH precursors in plasma. We have subsequently quantified the concentrations of ACTH precursors in the human circulation under basal conditions. Direct measurement of these peptides was made possible by the development of a two-site immunoradiometric assay for the ACTH precursors, POMC and pro-ACTH (Crosby et al., 1988). The assay is based on a labelled monoclonal antibody which binds within the ACTH region of POMC and a solid phase antibody which recognizes N-terminal POMC (N-POC, Fig. 1). As binding of both antibodies is required to generate a signal, the assay does not detect ACTH. Using this assay, the concentrations of ACTH precursors in normal subjects were found to be 5-40 pmol/l which is equivalent to or greater than those of ACTH, N-POC, b-lipotrophin (b-LPH) and b-endorphin (Gibson et al., 1994). The levels of ACTH precursors and ACTH in the circulation at any given time are dependent on regulatory mechanisms influencing expression of the POMC gene. However, the relative concentrations of ACTH precursors and ACTH will depend on precursor processing and mechanisms of secretion from the corticotroph cells. Evidence from studies with the mouse corticotroph adenoma cell line, AtT20, suggests that in the absence of stimulation, corticotroph cells ‘leak’ newly synthesized POMC (Kelly, 1985). Furthermore, results we have obtained in normal subjects and from clinical studies indicate that acute regulation of secretion of ACTH and ACTH precursors differs (see below).

ACTH-like bioactivity and immunoactivity in fetal lamb pituitaries at 0.65 and 0.95 gestation.

We wished to determine if the concentration of bioactive ACTH-like activity increased during development and if there was heterogeneity in ovine fetal anterior pituitary ACTH activity as measured by bioassay and radioimmunoassay (RIA). We obtained anterior pituitaries from eight sheep fetuses (four at 0.65 and four at 0.95 gestation; term 145 +/- 5 days) and extracted and homogenized them in ice-cold 5N acetic acid, 0.3% phenylmethanesulfonyl fluoride (PMSF) and 0.2% BSA. Fractionation of each pituitary extract was performed by size-exclusion chromatography using Sepadex G-50. The ACTH-like immunoactivity (ALI) profile for each pituitary showed two well-defined peaks. One eluted with human ACTH1-39 and the other eluted with the high molecular weight fraction in the void volume. Four fractions from the first peak representing the high molecular weight forms of ACTH activity and four fractions from the second peak representing the low molecular weight forms of ACTH activity were pooled separately. These two pools were subjected to reverse-phase chromatography (RPC) on a C-8 column using a linear gradient of 70% acetonitrile in 0.8% trifluoroacetic acid over a 60 min period. Based upon the RIA, the high molecular weight forms of ACTH from the G-50 column were resolved into three main fractions, one eluting similar to the standard ACTH1-39 and the remaining two eluting after that. The low molecular weight forms of ACTH from the G-50 column were resolved into three peaks, before, with, and after the standard. We used collagenase-dispersed rat adrenal cells to test the ACTH-like bioactivity (ALB) of the crude extracts and of the different fractions obtained from the RPC of the high and low molecular weight material. The concentration of ACTH-like bioactivity in the crude extracts was similar at the two stages of gestation. However, there was a trend for the low molecular weight peak to have more peptide eluting with human ACTH1-39 and higher ratios of ALB/ALI than did the high molecular weight peak. These results suggest that multiple ACTH molecular forms with different ALB/ALI ratios are present in the ovine fetal pituitary and that there is no selective increase in ACTH1-39 concentration in the fetal pituitary in late gestation.

Brain acth prevents stress gastric lesions in rats

This study evaluated the effect of ACTH and several ACTH fragments on the development of gastric glandular lesions induced by cold-restraint stress in rats. Intracerebroventricular administration of ACTH 1–39 dose-dependently (0.1–10 μg) inhibited stress gastric lesion formation. Studies with smaller molecular weight forms of ACTH (in a dose equimolar to 10 μg of ACTH 1–39) revealed that ACTH 1–13 and ACTH 1–10 were also protective. The ACTH fragments ACTH 5–10, ACTH 34–39 and ACTH 1–17 were without effect. Immunoneutralization of endogenous brain ACTH 1–39 significantly increased stress gastric lesion severity. Antisera raised against synthetic somatostatin, gonadotropin-releasing hormone, and L-enkephalin were ineffective. These results with ACTH coupled with our previous demonstration of a protective effect of β-endorphin suggest that specific brain pro-opiomelanocortin gene products modulate gastric mucosal integrity in response to stress. Brain ACTH ACTH fragments Stress ulcers ACTH antiserum Male rats

Gel chromatographic characterization of immunoreactive adrenocorticotropin in patients with ACTH hypersecretion

We investigated the molecular size of circulating immunoreactive ACTH by gel chromatography in patients with ACTH hypersecretion due to various disorders of the hypothalamic-pituitary-adrenal axis. 4 patients with Addison's disease, 2 with Nelson's syndrome, 4 with Cushing's disease, 6 with the ectopic ACTH syndrome (2 bronchial carcinoma, 1 medullary carcinoma, 1 metastatic islett cell carcinoma, 1 benign bronchial carcinoid and 1 patient with occult ectopic Cushing's syndrome) and 1 patient with hypersecretion of ACTH from a clinically nonfunctioning pituitary adenoma were studied. Analysis of the molecular size of immunoreactive ACTH was performed by gel chromatography on a Sephadex G-75 column (superfine, 100 x 1.5 cm) equilibrated with 1% formic acid. 2 ml fractions were collected and evaporated to dryness. The ACTH content of the recovered samples was determined by RIA. In Addison's disease, Nelson's syndrome and Cushing's disease the plasma showed a single peak of ACTH immunoreactivity at the expected position of 1-39 ACTH. In the ectopic ACTH syndrome the plasma of 4 patients revealed at chromatography at least one other peak eluting between the void volume and 1-39 ACTH suggestive of a high molecular weight form of ACTH whereas plasma of 2 patients showed only a single ACTH peak at the position of labeled 1-39 ACTH. The patient with a clinically non-functioning pituitary adenoma revealed a gel filtration pattern similar to the patients with ectopic ACTH syndrome and secretion of high molecular weight ACTH. We conclude that secretion of high molecular weight forms of ACTH is not a unique feature of the ectopic ACTH syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypersecretion of a large molecular weight form of ACTH by a clinically silent pituitary adenoma

Hypersecretion of a large molecular weight form of ACTH by a clinically silent pituitary adenoma

Changes in distribution of molecular weight forms of biologically active and immunoreactive adrenocorticotropic hormone after adrenalectomy in rat anterior pituitary.

The concentration of ACTH in extracts of rat anterior pituitary was measured by both radioimmunoassay and bioassay at different stages following adrenalectomy. Both types of ACTH activity decreased the day immediately following adrenalectomy but increased gradually afterwards. Immunological ACTH activity increased to 250% of the control value and biological ACTH activity increased to 490% of control value 3 weeks after adrenalectomy. The increase in biological ACTH activity occurred earlier, and the rate of increase was greater, than that of the immunological ACTH activity. The distributions of molecular weight forms of ACTH in extracts of anterior pituitary lobes was determined by gel filtration. Three molecular weight forms of immunoassayable ACTH were detected. Biological ACTH activity appeared in the 2nd and the 3rd peaks. A striking change was observed after adrenalectomy in the distribution of biologically active forms of ACTH. The ratio of biological ACTH activity to immunological ACTH activity in each peak changed at various stages after adrenalectomy. This indicated the heterogenous nature of the ACTH included in each peak. At 2 and again at 3 weeks, biological activity markedly increased until it exceeded the immunological ACTH activity in the 2nd peak. Dexamethasone had little influence on the elution profile of either immunoassayable and biologically active ACTH in gel filtration. Adrenalectomy may possibly have an effect on the intracellular posttranslational processing of ACTH precursors which leads to the development of biological ACTH activity.

Plasma pattern of immunoreactive ACTH in normal man and in patients with Nelson's syndrome.

A radioimmunoassay for ACTH determinations in human plasma was developed using an N-terminal antiserum. Plasma ACTH levels at 6 a.m. in 39 patients with normal pituitary-adrenal function were below 4 pmol/l in two individuals. The remaining had a mean of 32.7 +/- 4 pmol/l (n = 37). Gel filtration on Sephadex G-50 medium and G-75 revealed that a high molecular weight immunoreactive ACTH was found in plasma from both normal individuals and patients with Nelson's syndrome. The stimulation of ACTH secretion with metyrapone (750 mg perorally) significantly increased the mean plasma levels of 1-39 ACTH after 3 h (from 0.9 to 6.6 pmol/l) in 10 healthy individuals. The level of the high molecular weight form remained constant. In one patient with Nelson's syndrome hydrocortisone iv (100 mg/h for 3 h) suppressed the total ACTH levels from 360 to 83 pmol/l following corticoid withdrawal on the previous evening, but did not affect the high molecular weight form of ACTH. The difference between the amounts of immunoreactive ACTH found in plasma from normal individuals and from patients with Nelson's syndrome was attributed to 1-39 ACTH.

Forms and activity of high molecular weight adrenocorticotropin in guinea pig

High molecular weight forms of adrenocorticotropin (ACTH) and endorphin were identified in extracts of guinea pig anterior and intermediate/posterior pituitary. Extracts of anterior pituitary contained ACTH immunoactive material with apparent molecular weights of 36,000, 24,000 and 4,500 daltons. The highest molecular weight form of ACTH co-migrated with a peak of endorphin immunoactive material. No material the size of glycosylated ACTH(1–39) was detected. Separated forms of high molecular weight ACTH prepared from mouse tumor cell culture medium stimulated the same maximal production of steroid as ACTH(1–39) in the guinea pig adrenal cell bioassay. Pro-ACTH/endorphin and ACTH biosynthetic intermediate were two orders of magnitude less potent than synthetic human ACTH(1–39); glycosylated ACTH(1–39) was equipotent to ACTH(1–39) although no similar material was detected in guinea pig pituitary extracts. Isolated guinea pig adrenal cortical cells were incubated with the various separated forms of mouse tumor cell ACTH and products synthesized from ( 3H)pregnenolone were analyzed by two-dimensional thin-layer chromatography. The ratio of cortisol-related to corticosterone-related products was the same in response in glycosylated and nonglycosylated ACTH.

Secretion of ACTH, LPH and beta-endophin from human pituitary tumours in vitro.

Basal and stimulated secretion of immunoreactive ACTH, LPH and beta-endorphin from four human pituitary tumours has been studied in vitro using a superfused, isolated cell system. Chromatography of cell secretions under acid-dissociating conditions demonstrated that the human tumor cells released immunoreactive peptides with the elution profiles of alpha h (1-39) ACTH, beta h-LPH, gamma h-LPH and beta h-endorphin confirming that beta h-endorphin is secreted by human pituitary tumour cells and is not formed by enzymic cleavage from beta h-LPH in blood. No alpha- or beta h-MSH, nor any higher molecular weight forms of ACTH or LPH were detected. The cells from all four tumours responded to stimulation with rat stalk-median eminence extract (SME) and synthetic AVP with a concomitant release of ACTH, beta-LPH, gamma-LPH and beta-endorphin. In contrast to the isolated rat anterior pituitary cells, the pattern of responses to SME and AVP were indistinguishable and the release provoked by rat SME could be accounted for virtually entirely by its vasopressin content. No stimulation of release was observed when the cells were exposed to a variety of biogenic amines. Addition of hydrocortisone to the perfusion buffer of two tumours resulted in a slow inhibition of both basal and stimulated ACTH and LPH release. These data demonstrate that human pituitary tumour tissue from patients with Cushing's disease and Nelson's syndrome can be studied in vitro and that such studies may contribute to a greater understanding of the aetiology of these diseases.

In vitro effects of high molecular weight forms of ACTH on the fetal sheep adrenal.

The direct involvement of the pituitary-adrenal axis in birth has been well established, at least in sheep, and its removal prolongs pregnancy. As part of the process the fetal sheep adrenal grows rapidly during the 10-15 d prepartum and is associated with a large rise in the plasma corticosteroid concentration. This does not seem to result from an increased ACTH secretion. The fetal adrenal in vivo seems refractory to circulating ACTH and shows poor response to elevation of plasma concentration. Thus the signal for the adrenal hypertrophy and the initiation of parturition remains unclear. The responsiveness of the fetal adrenal to ACTH has been re-examined using isolated adrenal cells. The study shows that in the fetal sheep these are not inherently unresponsive to ACTH, but that high-molecular-weight forms of ACTH block the action of ACTH. These peptides may be responsible for controlling the activity of the adrenal in situ.

Synthesis and secretion of corticotropins, melanotropins, and endorphins by rat intermediate pituitary cells.

The synthesis and secretion of various intermediate pituitary proteins was studied by using dispersed intermediate pituitary cell suspensions. Control studies indicated that the isolated cells were obtained in good yield and that after more than 24 h in culture the isolated cells continued to synthesize a collection of proteins similar to those found in freshly extracted intermediate pituitary tissue. Rat intermediate pituitary cells synthesized a molecule (Mr = 30,000; called 30K) that contained antigenic determinants for beta-endorphin, gamma-lipotropin, corticotropin (ACTH), and 16K fragment (the NH2-terminal region of mouse tumor cell pro-ACTH/endorphin). This 30K molecule, two high molecular weight forms of ACTH(13K and 20K), and 16K fragment were all shown to be glycoproteins. Continuous labeling and pulse-chase incubations were used to define the intracellular biosynthetic processing of the 30K molecule. After a 15-min pulse incubation the 30K molecule was the only labeled protein containing antigenic determinants for beta-endorphin, gamma-lipotropin, ACTH, or 16K fragment. A beta-lipotropin-like molecule served as a biosynthetic intermediate in the production of proteins similar to beta-endorphin and gamma-lipotropin. Methionine-enkephalin and alpha-endorphin were not major products in the intermediate lobe cells. Molecules similar to alpha-melanocyte-stimulating hormone and corticotropin-like intermediate lobe peptide (ACTH(18-39)) were also derived from the same 30K molecule; 20K ACTH served as a biosynthetic intermediate in this conversion. In rat intermediate pituitary cells ACTH(1-39) was not a major final product of the intracellular biosynthetic processing of the 30K molecule. The 30K molecule also served as a precursor to a protein similar to mouse tumor cell 16K fragment and related smaller proteins. With rat intermediate pituitary cells, pulse-chase experiments utilizing [35S]methionine demonstrated almost quantitative conversion of the 30K precursor into labeled proteins similar to beta-endorphin and alpha-melanocyte-stimulating hormone. In the absence of added secretagogues, small amounts of all of the smaller proteins derived from the 30K precursor were secreted coordinately into the culture medium.

Molecular Weight Forms of Adrenocorticotropic Hormone Secreted by Primary Cultures of Mouse Anterior Pituitary*

Extracts of mouse anterior pituitary cells in monolayer culture were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to separate the molecular weight forms of ACTH. The gels were sliced and each segment was eluted. The eluates were assayed for ACTH immunoactivity. Approximately 10% of the immunoactivity in the extracts was found to be present as 20,000--32,000 mol wt ACTH. The remainder of the immunoactivity was equally distributed between two forms of ACTH with apparent molecular weights of 11,800 and 4,500. This distribution is very similar to that found in extracts of mouse anterior pituitary. Mouse anterior pituitary cultures were incubated for 2 h in serum-free tissue culture medium. ACTH was concentrated from the medium and fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The medium was found to contain predominantly the 4,500 and 11,800 forms of ACTH. When vasopressin was added to these cultures (100 ng/ml), the rate of secretion of ACTH was more than doubled in serum free medium. Analysis of the medium from vasopressin-stimulated cultures showed that the 4,500 and 11,800 mol wt forms of ACTH were again the predominant forms present.

Coordinate control of corticotropin, β-lipotropin, and β-endorphin release in mouse pituitary cell cultures

Hypothalamic extract stimulates the release of corticotropin (ACTH) and endorphins 2.5- to 30-fold in mouse pituitary tumor cell cultures (AtT-20/D(16v) line) and primary cell cultures from mouse anterior pituitary. ACTH and endorphin activities were measured by radioimmunoassay and immunoprecipitation. Pretreatment of tumor cell cultures with 1 muM dexamethasone reduced the stimulatory effect of the extract on release of ACTH and endorphins. Pretreatment of primary cell cultures with 10(-6) M dexamethasone reduced the stimulatory effect of both vasopressin and the extract on the release of ACTH and endorphins. Release of ACTH and endorphin was coupled in both kinds of cultures in the basal, stimulated, and inhibited states. The molecular weight forms of ACTH and endorphin in tumor cell culture medium were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Radioimmunoassay and immunoprecipitation show that the 13,000-dalton and 4500-dalton forms of ACTH were present in about equal amounts in medium from cultures incubated with or without hypothalamic extract for 15 min, 30 min, or 2 hr. Smaller amounts of the high molecular weight forms of ACTH (20,000- to 23,000-dalton and 31,000-dalton ACTH) were observed in the culture medium at these times. The predominant forms of endorphin released after 20 min or 3 hr of incubation had molecular weights of 31,000, 11,700 (beta-lipotropic hormone-size material) and 3500 (beta-endorphin-size material). No degradation of the forms of endorphin released into the culture medium was observed after incubating the culture medium for 1.5 hr in the absence of cells. The proportions of the different forms of endorphin and ACTH present in the culture medium resembles that seen in cell extracts.

Steps involved in the processing of common precursor forms of adrenocorticotropin and endorphin in cultures of mouse pituitary cells

The initial steps in the processing of the common precursor to adrenocorticotropin (ACTH) and endorphin in mouse pituitary tumor cells (AtT-20) have been investigated. Three forms of the precursor have been resolved by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis with apparent molecular weights of 29 000 (29K ACTH-endorphin), 32 000 (32K ACTH-endorphin) and 34 000 (34K ACTH-endorphin). These forms have a similar peptide backbone, but their carbohydrate content differs. In particular, a tryptic glycopeptide has been observed in 32K ACTH-endorphin which is not present in 29K ACTH-endorphin and has been identified as the tryptic peptide containing the alpha(22--39) sequence of ACTH. Similar heterogeneity in carbohydrate has been observed in some of the smaller molecular weight forms of ACTH which are resolved by NaDodSO4 gel electrophoresis. Pulse chase and continuous labeling studies using radioactive amino acids and sugars suggest that the 29K ACTH-endorphin is converted to 32K and 34K ACTH-endorphin by the addition of carbohydrate. The glycopeptide and pulse chase studies suggest that 29K ACTH-endorphin is at a branch point in the processing pathways. It can either be converted to 4.5K ACTH by proteolytic processing or to 32K ACTH-endorphin by the further addition of carbohydrate. The 32K ACTH-endorphin can then be converted to 13K ACTH, the glycosylated form of 4.5K ACTH (Eipper, B.A., & Mains,, R.E. (1977) J.Biol. Chem.252, 882), by proteolytic processing. A comparison of the distribution of the different molecular weight forms of ACTH and endorphin in mouse pituitary extracts and in the mouse pituitary tumor cells reveals that the pituitary contains all of the forms of ACTH and endorphin seen in the tumor cells, including the three forms of the ACTH-endorphin precursor. However, the molecular weight distribution of the forms in the anterior lobe is very different from that in the intermediate lobe of mouse pituitary.

High molecular weight forms of adrenocorticotropic hormone are glycoproteins.

Mouse pituitary tumor cells (AtT-20/D-16v) were incubated in medium containing [3H] glucosamine or [3H] mannose. By analyzing immunoprecipitates of cell extracts and culture medium it was shown that [3H] glucosamine and [3H] mannose were incorporated into all three high molecular weight forms of ACTH; label was not incorporated into Mr=4,500 ACTH (which is thought to be similar to the 39 amino acid polypeptide form of ACTH, alpha(1-39)). Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis the apparent molecular weights of these glycoprotein forms of ACTH were 31,000, 23,000, and 13,000. Gel filtration in 6 M guanidine HCl indicated that the molecular weights of these forms of ACTH were substantially lower; sodium dodecyl sulfate-polyacrylamide gel electrophoresis has often been found to overestimate the molecular weight of glycoproteins. A significant fraction of the high molecular weight ACTH in tumor cell extracts binds to columns of concanavalin A-agarose and can be eluted with 0.2 M alpha-methyl-D-mannopyranoside; porcine alpha(1-39) does not bind to concanavalin A-agarose. High molecular weight glycoprotein ACTH can be detected in extracts of mouse and bovine pituitary by using concavalin A affinity chromatography.