ACTH Immunoactivity Research Articles

ACTH immunoactivity in normal rat tissues: modulation by hypophysectomy and adrenalectomy.

This study investigates the effect of pertubation of the normal pituitary-adrenal axis on concentrations of adrenocorticotropin (ACTH)-like immunoactivity in peripheral tissues. We used a polyclonal antibody (West antibody) to measure ACTH-like immunoactivity in glacial acetic acid extracts of five tissues in adult male rats at increasing times (1, 7, 14, and 28 days) after hypophysectomy or adrenalectomy, and in normal control rats. Concentrations of ACTH-like immunoactivity were similar to those previously reported in liver, colon, heart, and small intestine and were not significantly affected by either hypophysectomy or adrenalectomy. While hypophysectomy also had no effect in the kidney, adrenalectomy resulted in a four-fold increase in extractable immunoactivity, first noticeable at seven days (p less than 0.005), but increasing progressively to 28 days (p less than 0.0005). Gel filtration showed that most of the increase in activity in kidneys of adrenalectomized rats corresponded to the 4.5 kD form comprising most of the serum ACTH immunoactivity and suggesting that the activity increase in kidney was largely due to ACTH derived from blood.

Large molecular weight ACTH immunoactivity in a patient with adrenoleukomyoloneuropathy

Very high ACTH immunoactivity was measured in a patient with adrenoleukomyeloneuropathy. The basal adrenocortical function was hyperactive, whereas the response to exogenous ACTH was depressed. Most of the ACTH immunoactivity was a large molecule (55,000 daltons), which weakly stimulated steroidogenesis of adrenocortical cells in vitro, but inhibited the binding of 125I-iodoACTH to the cells and suppressed ACTH-induced steroid production. Abnormalities in the pituitary-adrenal axis in adrenoleukomyeloneuropathy have been considered to be solely attributable to destruction of the adrenal cortex. In the current case, however, large molecular weight ACTH immunoactivity is present and might have some role in the adrenocortical dysfunction.

Partial purification of an adrenal growth factor produced by normal bovine anterior pituitary cells in culture.

An adrenal growth factor (AGF) was partially purified from the conditioned medium of cultured bovine anterior pituitary cells. This 900-fold purification was accomplished by gel filtration, ammonium acetate precipitation, cation exchange fast protein liquid chromatography, and C8 reverse phase fast protein liquid chromatography. The AGF had an apparent mol wt of 25,000-30,000, as determined by gel filtration chromatography at acid pH. It was tested on mouse fibroblasts (3T3 cells), rat fibroblasts (NRK cells), mouse adrenocortical cells (Y1 cells), and normal bovine adrenocortical cells (BAC cells) and found to be a relatively potent mitogen for BAC cells only, contrasting with fibroblast growth factor, which could stimulate all of these cell types. It is sensitive to reducing agent, alkaline pH, and boiling. Purification steps were carried out at acid pH, suggesting some degree of stability to acidic pH. The mitogenic activity in the conditioned medium could be blocked by ACTH, and the purified AGF contained no ACTH immunoactivity. The AGF appears distinct from epidermal growth factor, since it could not bind to epidermal growth factor receptors, and platelet-derived growth factor, which could not stimulate BAC cells. Purification to homogeneity was not accomplished, because very broad bands of mitogenic activity were observed with all of the chromatographic methods tried. The AGF appears to be distinct from any of the known growth factors.

Clinical evaluation of a two-site immunoradiometric assay for adrenocorticotrophin in unextracted human plasma using monoclonal antibodies.

We have developed a sensitive two-site immunoradiometric assay (IRMA) for intact ACTH and its precursors, pro-opiomelanocortin and 22 kDa peptide in unextracted human plasma. The assay uses two monoclonal antibodies. Antibody 1A12, specific for ACTH 10-18, is radiolabelled and antibody 2A3 specific for the C-terminal region (ACTH 24-39), is coupled to Sephacryl S300 for the solid-phase. Samples are incubated for 18 h with labelled antibody followed by 2 h with solid-phase antibody. Separation employs the sucrose layering technique. Using human pituitary ACTH 1-39 (code 74/555) in diluent containing 10% horse serum to standardize the assay, the sensitivity (upper 99% confidence limit of zero standard) is 3.5 +/- 0.8 ng/l (n - 7). The mean coefficient of variation is 5.9% within-assay and 6.7% between-assay and is less than 10% between 22 and greater than 5000 ng/l. Mean recovery of ACTH 1-39 added to dexamethasone-suppressed human plasma is 109% and endogenous ACTH behaves indistinguishably from standard ACTH on dilution. In normal subjects, mean plasma ACTH levels are 30 ng/l at 0730 h, and 15 ng/l at 1630 h at rest. ACTH concentrations are between 60 and 330 ng/l, 8-10.5 h after metyrapone (2 g orally at 2300 h), between 140 and 320 ng/l, 30-60 min after insulin-induced hypoglycaemia, and less than 4 ng/l, 8 h after dexamethasone (1.5 mg orally at 2300 h). In a range of pathological conditions ACTH concentrations accurately reflect the disorders of the pituitary-adrenal axis. Endogenous ACTH immunoactivity is stable in vitro at 22 degrees C for at least 1 h in whole blood and at least 4 h in plasma. It is concluded that this two-site IRMA for ACTH in unextracted plasma offers a reliable assay for clinical purposes.

Adrenocorticotropin immunoactivity in monkey hypophyseal portal blood.

ACTH was measured with both C-terminal and midportion antibodies in monkey hypophyseal portal plasma, and compared to levels in monkey peripheral plasma, medial basal hypothalamus, and anterior pituitary. In nine female monkeys, mean hypophyseal portal blood C-terminal ACTH immunoactivity was 5290 +/- 2010 (SEM) pg/ml, whereas the mean midportion ACTH level was 949 +/- 178 pg/ml. These immunoactivities were not lower in two monkeys that were completely hypophysectomized 30 min before portal blood collection. The ratio of C-terminal to midportion ACTH immunoactivity was 4:1 in two monkey medial basal hypothalami, and 1:1 in four monkey anterior pituitary glands. Gel filtration of hypophyseal portal plasma extract and of medial basal hypothalamus showed that the C-terminal ACTH immunoactivity eluted in the same position as the corticotropin-like-intermediate lobe peptide standard. The similarity of the C-terminal to midportion ACTH ratios in monkey medial basal hypothalamus and portal blood, and the observation that ACTH immunoactivity was not significantly lower in two hypophysectomized monkeys suggests that portal blood C-terminal ACTH immunoactivity is of hypothalamic rather than pituitary origin. We conclude that monkey hypophyseal portal blood contains high levels of a C-terminal fragment of ACTH, which coelutes with corticotropin-like intermediate lobe peptide on gel filtration, and which is secreted from the brain directly into the portal circulation.

Widespread presence of large molecular weight adrenocorticotropin-like substances in normal rat extrapituitary tissues.

ACTH activity in glacial acetic acid extracts of normal rat tissues was studied by both ACTH RIA and a sensitive in vitro bioassay. ACTH immunoactivity was found in all tissues: brain, 278 +/- 54 (mean +/- SE; picograms per mg protein); stomach, 59 +/- 4; kidney, 47 +/- 3; colon, 40 +/- 4; small intestine, 37 +/- 4; liver, 18 +/- 2; and heart, 16 +/- 3. Tissue ACTH showed parallelism with ACTH standard in the RIA. No correlation existed between tissue ACTH and plasma ACTH in normal rats. Dexamethasone treatment (0.4 mg/day for 5 days) suppressed plasma ACTH, but did not affect tissue ACTH levels. When tissue extracts were passed through Sephadex G-75-SF columns, ACTH immunoactivity was exclusively eluted in the portion of bigger molecular weight than ACTH standard, except in the brain. Based on this column chromatography, the molecular weight of the main peak of activity was estimated as 26,000. Tissue ACTH-like material contained no detectable biological activity (less than 2 pg/100 ng tissue). However, biological activity was easily detectable after exposure of the tissue extracts to trypsin. When studied by immunoassay and bioassay, this 26,000 mol wt ACTH was digested and cleaved to 4,500 mol wt and biologically active ACTH with trypsin treatment. Tissue ACTH immunoactivity does not seem to be the result of artifacts: 1) extracts were adjusted to pH 8.0 and a common osmolality (150 mosmol/liter) before assay; 2) protein contents in RIA tubes were only 0.1-1.6 mg; 3) tissue extracts incubated with [125I]iodo-ACTH for 48 h produced less than 5% damage of labeled hormone, as assessed by moderate excess of antibody binding; 4) enzyme inhibitors did not modify tissue ACTH levels; and 5) ACTH immunoactivity of tissue extracts was absorbed by anti-ACTH immunocolumns. We conclude that high molecular weight ACTH-like materials are widespread in normal rat extrapituitary tissues and are probably a precursor form of 4500 mol wt ACTH.

Correlation between electrical activity and ACTH/beta-endorphin secretion in mouse pituitary tumor cells.

The electrical and secretory activities of mouse pituitary tumor cells (AtT-20/D-16v), which contain and release the ACTH/beta-endorphin family of peptides, were studied by means of intracellular recordings and radioimmunoassays. Injection of depolarizing current pulses evoked action potentials in all cells and the majority (82%) displayed spontaneous action potential activity. Action potentials were found to be calcium-dependent. Barium increased membrane resistance, action potential amplitude and duration, and release of ACTH and beta-endorphin immunoactivity. Isoproterenol increased both action potential frequency and hormone secretion. Raising the external calcium concentration increased the frequency and amplitude of the action potentials and stimulated secretion of ACTH and beta-endorphin immunoactivity. Thus, stimulation of secretory activity in AtT-20 cells was closely correlated with increased electrical activity. However, a complete blockade of action potential activity had no effect on basal hormone secretion in these cells. These results suggest that the mechanisms underlying stimulated hormone secretion are different from those responsible for basal secretory activity. It is proposed that the increased influx of calcium due to the increased action potential frequency initiates the stimulated release of hormone from these cells.

Circulating ACTH and related peptides in lung cancer.

The prevalence of high levels of circulating ACTH-like immunoactivity was determined in 134 patients with lung cancer, using reference ranges from 52 age- and sex-matched patients with non-malignant lung disease. Two studies used ACTH radioimmunoassays with different specificities. Study A used an unextracted plasma or serum assay for total ACTH immunoactivity. High serum ACTH levels occurred in 24% of patients with small-cell carcinoma and 3% of patients with non-small-cell cancer. In patients with small-cell carcinoma, levels were high in 12% with limited disease and 32% with extensive disease. Study B used an ACTH assay after plasma extraction by porous glass, which measured mainly regular 1-39 ACTH. Here no lung-cancer patient had levels above the reference range, suggesting that the high levels in Study A may be due to plasma ACTH components which are poorly extracted by porous glass. It is concluded that high circulating ACTH immunoactivity occurs in a minority of patients with lung cancer, particularly those with extensive small-cell carcinoma. Indirect evidence suggests that the high ACTH levels detected with assays for total ACTH are due to molecular forms other than 1-39 ACTH, probably high-mol.-wt species.

The distribution of forms of adrenocorticotropin and beta-endorphin in normal, tumorous, and autopsy human anterior pituiary tissue: virtual absence of 13K adrenocorticotropin.

To begin to define the nature of the biosynthesis and processing of ACTH and beta-endorphin in the human, anterior pituitary tissue (fresh normal and adenomatous, and autopsy) was extracted in acetic acid in the presence of protease inhibitors and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel slice eluates were assayed for ACTH and beta-endorphin immunoactivity. Human anterior pituitary tissue contained four major size classes of ACTH and three major size classes of beta-endorphin. We found that in all tissue sources examined there was a virtual absence of 13-15K ACTH, which is a major form in the rat and mouse. When comparing extracts obtained from fresh normal or adenomatous anterior pituitary tissue, we also found a drastic decrease in beta-lipotropin and beta-endorphin in extracts of autopsy human anterior pituitaries. These results suggest that the biosynthesis and processing of pituitary ACTH and beta-endorphin in the human may be different than in the mouse, and because of apparent postmortem proteolysis of beta-endorphin, human pituitary obtained at autopsy is probably not a good source of material for biochemical studies of pituitary tissue.

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.

LUNG TUMOURS AND ACTH PRODUCTION

ACTH levels measured by N- and C-terminal immunoassays and cytochemical bioassay, were measured in fourteen lung tumours not associated with the ectopic ACTH syndrome and in macroscopically normal lung tissue taken from the same patients at thoracotomy. Significant concentrations of immunocative (greater than 3 ng/g wet weight) and bioactive (greater than 0.2 ng/g wet weight) ACTH were found in all the carcinoid and oat cell tumours (n=9), a combined tumour (poorly differentiated adenocarcinoma with large cell carcinoid elements), and a poorly differentiated squamous cell tumour. All the carcinoid tumours contained more C- than N-terminal ACTH immunoactivity. The squamous cell tumours (n=2), anaplastic tumours (n=2) and adenocarcinoma contained insignificant ACTH levels. There was a good correlation between the ACTH levels and the presence of secretory granules in the tumours examined ultrastructually. All the macroscopically normal samples of lung tissue contained immuno and bioactive ACTH-like material, the levels of which correlated well with ACTH levels in the tumour tissue. It is suggested that all lung tumours of carcinoid or oat cell type synthesize ACTH-like materials although clinical evidence of the ectopic ACTH syndrome may be absent. The presence of ACTH-like materials in non-tumorous lung tissue in patients with lung cancer may indicate a low level of ACTH production throughout the lung or sequestration of ACTH containing granules secreted by the tumour.