Corticotrophin-releasing factor-like immunoreactivity and bioactivity of human fetal and adult hypothalami.

Corticotrophin releasing factor-like immunoreactivity (CRF-LI) and bioactivity, and arginine vasopressin-like immunoreactivity (AVP-LI) have been measured in extracts of human fetal and adult hypothalamic tissue and their development with the gestational age of the fetuses (12-27 weeks) studied. CRF-LI was measured by a radioimmunoassay developed for ovine corticotrophin-releasing factor (oCRF-41). Corticotrophin-releasing factor bioactivity was measured in a rat isolated anterior pituitary cell perfusion system. CRF-LI and bioactivity and AVP-LI were all detectable in fetal hypothalamic extracts from 12 to 13 weeks of gestational age. CRF-LI was also present in human fetal pituitary glands from 12 weeks of gestational age. The concentration of CRF-LI in the fetal hypothalamic extracts (9.2 +/- 11.4 ng/g, mean +/- S.E.M., n = 33) showed no significant correlation with the gestational age of the fetuses. However the concentration of AVP-LI (25.0-36.8 ng/g, n = 17) did show a positive correlation (r = 0.508, P less than 0.05) with gestational age, as did the concentration of CRF bioactivity (471.3-556.3 ng ACTH released/g tissue, n = 13, r = 0.725, P less than 0.01). The CRF bioactivity of all fetal hypothalamic extracts was potentiated by the addition of synthetic human (h)AVP, but the bioactivity of the adult hypothalamic extracts was not, presumably because of the higher levels of AVP-LI already present in the adult extracts. Pretreatment of tissue extracts with antisera to oCRF-41 and/or hAVP reduced the CRF bioactivity of all hypothalamic extracts. Sephadex chromatography of fractions which co-eluted with synthetic oCRF-41 or hAVP contained CRF bioactivity and this bioactivity was potentiated when synthetic hAVP or oCRF-41, respectively, were added to the fractions. However, a larger molecular weight form of CRF-LI (8000-10 000 daltons), which was observed only in fetuses of 20 weeks of gestational age or less, did not contain any significant CRF bioactivity.

A case of pituitary dependent Cushing's disease with clinical and biochemical features of the ectopic ACTH syndrome.

A case of atypical pituitary dependent Cushing's disease is reported. The patient presented with clinical symptoms similar to those of the ectopic ACTH syndrome; notably a marked hypokalaemic alkalosis, widely fluctuating plasma cortisol levels, greatly elevated plasma ACTH levels, and failure to suppress both plasma cortisol and ACTH levels following high dose oral dexamethasone. However, a large aggressive pituitary tumour was detected by skull X-ray and computed tomography. Removal of the pituitary tumour led to full remission of the patient's Cushing's syndrome. Pro-opiomelanocortin (POMC) related peptides in the plasma and tumour tissue extract of this patient have been characterized by gel-filtration and Concanavalin-A Sepharose affinity chromatography, indicating processing of POMC in a manner more usually associated with ectopic tumours.

Measurement of immunoreactive gamma-MSH in human plasma.

A radioimmunoassay for immunoreactive gamma-MSH (IR-gamma-MSH) in human plasma has been developed. The assay is capable of detecting normal basal circulating levels which range from less than 20-100 ng/1 at 0900 h. Plasma levels are raised concomitantly with ACTH during insulin induced hypoglycaemia and CRF stimulation and suppressed with dexamethasone. Chromatographic characterisation of IR-gamma-MSH in plasma demonstrates a major peak of IR-gamma-MSH, corresponding to purified glycosylated N-terminal pro-opiomelanocortin 1-76, when IR-gamma-MSH is secreted from the pituitary. In contrast IR-gamma-MSH produced ectopically appears to be heterogeneous.

Corticotrophin-releasing factor--mediated adrenocorticotrophin release from rat anterior pituitary cells is potentiated by C-terminal gastrin-releasing peptide.

The remission of Cushing's syndrome following surgical removal of a tumour containing bombesin-like immunoreactivity (BLI), but insignificant levels of ACTH, is described. However, an acid extract of the tumour tissue caused the release of ACTH from isolated rat anterior pituitary cells in vitro. These observations led to an investigation of the effects of synthetic C-terminal gastrin-releasing peptide (GRP(14-27] on ACTH release from isolated rat anterior pituitary cells. GRP(14-27) (10-1000 ng/ml) directly stimulated the release of ACTH in vitro, whereas lower doses (10-1000 pg/ml), ineffective themselves in eliciting ACTH release, potentiated the CRF-mediated in-vitro release of ACTH.

Regional distribution of pro-opiomelanocortin-derived peptides in the human brain.

The regional distribution of pro-opiocortin-derived peptides and methionine enkephalin was investigated in human brain post-mortem. Sequence-directed radioimmunoassays for beta-endorphin, gamma-lipotropin, adrenocorticotrophin, corticotrophin-like intermediate lobe peptide (ACTH18-39, CLIP) alpha-MSH and methionine enkephalin were used and 40 different human brain areas were assayed. The regional distribution of all the pro-opiomelanocortin-derived immunoreactivities were correlated with highest amounts of beta-endorphin, gamma-lipotropin and ACTH in the hypothalamus, amygdala, periventricular grey, substantia nigra and superior colliculus. The distribution of beta-endorphin, gamma-lipotropin and ACTH did not parallel the distribution of methionine-enkephalin immunoreactivity which was present in the globus pallidus, nucleus accumbens and substantia nigra. Gel exclusion chromatography (G-50) showed that pro-opiocortin-related peptides in the human hypothalamus and periventricular grey separated in positions consistent with the major immunoreactive forms being beta-endorphin, gamma-lipotropin, ACTH and CLIP.

Endogenous opioid peptides in pregnancy.

Plasma levels of two endogenous opioid peptides, beta-endorphin and met-enkephalin, as well as immunoreactive N-terminal beta-lipotrophin (N-LPH) were studied in normal pregnant women from 8 to 41 weeks gestation. A total of 116 samples were assayed for beta-endorphin-like immunoreactivity (C-LPH), 103 for N-LPH and 75 for met-enkephalin. Plasma beta-endorphin-like immunoreactivity and N-LPH levels rose progressively throughout gestation and reached a maximum at term. Plasma met-enkephalin immunoreactivity did not significantly change throughout pregnancy. These results reflect an increasing and consistent adrenocorticotrophin (ACTH)/beta-LPH and beta-endorphin secretion throughout pregnancy, while the unchanged met-enkephalin levels are compatible with its known derivation from a separate precursor system.

Pro-opiocortin related peptides in human pituitary and ectopic ACTH secreting tumours.

Basal and stimulated secretion of N-terminal pro-opiocortin (Pro-gamma-MSH), ACTH and LPH from seven pituitary and three ectopic ACTH secreting tumours have been studied in vitro using a perfused isolated cell system. The peptides were shown to be released concomitantly and in equimolar amounts. The pituitary tumours responded to stimulation with rat stalk median eminence extracts (SME) and synthetic AVP. However, peptide release from the ectopic tumours, although pulsatile, remained autonomous. Prior to surgery, gel-chromatographic profiles of plasma immunoreactive ACTH showed only one peak, which eluted in the position of 1-39 ACTH, in patients with the pituitary tumours, but there was a second peak of large molecular weight ACTH present in the plasma from those with the ectopic ACTH syndrome. This second form of ACTH could not be detected in any of the tumour cell column effluents. An eighth pituitary tumour was atypical, in its unusually large size, clinically aggressive nature and spectrum of peptide release. Although peptide release in response to stimulation with SME was similar to that observed with the other pituitary tumours, the chromatography of the plasma ACTH resembled the ectopic plasma pattern, showing two peaks of immunoreactivity.

Characterisation of immunoreactive somatostatin in human fetal hypothalamic tissue.

Levels of immunoreactive somatostatin (IRS) were measured in extracts of hypothalamic tissue from human fetuses of 12-26 weeks gestation. The IRS contents (0.7-22.5 ng) and concentrations (2.7-118.0 pg/mg wet weight tissue) both increased slightly with gestation. Sephadex G-50 chromatography of 11 extracts showed up to four peaks of IRS, one co-eluting with synthetic somatostatin-14 (S14), a second co-eluting with synthetic somatostatin-28 (S28) and two other peaks having approximate molecular weights of 6000 and 10 000, respectively. The levels of S14 and S28 increased significantly during gestation, while the levels of 6000 molecular weight IRS decreased with age. We suggest that the increase in S14 and S28 levels may be the cause of the fall in circulating growth hormone (GH) in the fetus in later gestation.

Stability of pro-opiocortin-related peptides in post-mortem mouse brain tissue.

The stability of pro-opiocortin-related peptides, growth hormone and somatostatin was investigated in mouse brain for up to 72 h post mortem. The peptide content in acid extracts of whole mouse brain was measured by radioimmunoassay and the molecular forms characterised by chromatography on Sephadex G-50 under acid-dissociating conditions. The brain content of the pro-opiocortin-related peptides and growth hormone rose markedly with time after death and chromatography showed this to be due to an increase in the same molecular forms present immediately post mortem, with no significant peptide fragmentation or precursor cleavage. These rises were not seen in the brains of those animals hypophysectomized at death. In contrast, the levels of somatostatin were not significantly altered post mortem though there was a shift in the relative distribution of immunoactivity between the different molecular forms. These results indicate that the peptide content of post-mortem mouse brain may be affected by leakage of pituitary contents and that even though the levels of other neuropeptides may not change, their molecular forms may be altered.

Experience with selective venous sampling in diagnosis of ACTH-dependent Cushing's syndrome.

Twenty-three patients with adrenocorticotrophic hormone-(ACTH)-dependent Cushing's syndrome were subjected to selective venous catheterisation and sampling for ACTH on a total of 26 occasions. Out of 10 patients with pituitary-dependent disease, nine had raised ACTH concentrations in one or both high internal jugular vein samples. Eight patients had 11 proved sites of ectopic hormone production: of these, six were correctly identified by the sampling technique, and in four of them this was the only accurate method of localisation. The results of one catheterisation were misleading, and on 10 occasions they were inconclusive; five patients remained undiagnosed by any method. Overall, 15 of the 26 catheterisations provided diagnostically valuable information. Selective venous catheterisation and sampling for ACTH is effective in confirming a pituitary source of the hormone and may be valuable in locating the source of ectopic ACTH production in some cases.

Development of a radioimmunoassay for an amino-terminal peptide of pro-opiocortin containing the gamma-MSH region: measurement and characterization in human plasma.

A radioimmunoassay has been developed for the N-terminal region of human pituitary pro-opiocortin (N-POC), the common precursor protein of ACTH and beta-LPH, using an antiserum which recognizes residues near the gamma-MSH region. The concentrations of greater than 300 ng/l of immunoreactive peptide were determined in unextracted human plasma, the relative molecular mass of the reacting fragments corresponding to a seventy-seven amino acid glycoprotein. The concentrations of immunoreactive N-POC peptides were correlated with those of ACTH in plasma obtained from patients with various disorders of the hypothalamic-pituitary-adrenal axis.

Characterisation of the pro opiocortin family of peptides in human cerebrospinal fluid.

Chromatography under acid dissociating conditions in conjunction with radioimmunoassay has been employed to investigate the nature of peptides related to opiocortin in human cerebrospinal fluid. Samples of cerebrospinal fluid (CSF) were collected for chromatography from 15 patients prior to air encephalography. 2 patients had pituitary dependent Cushing's disease, 3 non-endocrine neurological disease and 10 non-ACTH related pituitary disease. The column fractions were assayed for N- and C-terminal beta-lipotropin, N-terminal ACTH and gamma-MSH immunoreactivity. Elution profiles obtained from chromatography on Sephadex G-50 demonstrated peaks of immunoreactivity corresponding to the elution positions of synthetic human beta-endorphin, highly purified beta-lipotropin and highly purified gamma-lipotropin in all CSF samples. A peak of a large molecular weight material with N and C terminal beta-lipotropin immunoreactivity was also detected. Chromatography of CSF on Sephadex G-75 showed this large molecular weight peak to be comprised of peptides eluting in the positions of a 31K molecular weight marker with beta-lipotropin and ACTH immunoreactivity and a 16K molecular weight marker with gamma-MSH immunoreactivity. This suggests the presence of the common precursor to ACTH and LPH in the CSF.

Pars intermedia peptides: studies in adult humans.

A combination of radioimmunoassays and chromatography under acid-dissociating conditions has been used to obtain profiles of ACTH and LPH-related peptides in human plasma and cerebrospinal fluid. The spectra of peptides observed in these two fluids differ markedly. ACTH, beta-LPH, gamma-LPH and beta-endorphin are observed in the plasma of normal subjects and patients with increased pituitary ACTH secretion, whereas cerebrospinal fluid contains ACTH, beta-LPH, gamma-LPH and beta-endorphin, a 31 000-molecular-weight putative precursor having ACTH, LPH and gamma-MSH immunoreactivities, as well as pro-gamma-MSH(1-77) and smaller immunoreactive gamma-MSH fragments, alpha-MSH was not observed in blood or cerebrospinal fluid but this pars intermedia peptide and corticotropin-like intermediate lobe peptide (CLIP) were both found in tumour tissues obtained from patients with the ectopic ACTH syndrome. In vitro studies of human pituitary tumour tissues confirmed concomitant secretion of ACTH, beta-LPH, gamma-LPH, beta-endorphin and pro-gamma-MSH, which could be stimulated by a preparation of crude stalk median eminence and synthetic arginine vasopressin, from the rat, and could be suppressed by hydrocortisone. Clinical studies in which electroacupuncture was used to alleviate the symptoms of heroin withdrawal or recurrent pain revealed that concentrations of met-enkephalin and beta-endorphin, respectively, may rise in cerebrospinal fluid in association with relief of symptoms.

Adrenocorticotropin and lipotropin secretion by dispersed cell cultures of a human corticotropic adenoma: effect of hypothalamic extract, arginine vasopressin, hydrocortisone, and serotonin.

Basal and modulated secretion of ACTH and lipotropin (LPH) by cultures of trypsin-dispersed cells of a biopsy of a human corticotropic adenoma have been examined. ACTH secretion was detectable throughout the period of culture (13 days) but declined steadily from an initial production rate of 238 +/- 124 ng/3 X 10(5) cells/12 h. The time course of secretion showed a slower phase over the first 4 h, with increases up to 12 h. An extract of rat stalk median eminence caused a significant (P less than 0.005) dose-dependent increase in both ACTH and LPH secretion during 30 min. The patterns of response for ACTH and LPH were very similar; both exhibited a decline in the basal release of peptide subsequent to the period of stimulation. The addition of hydrocortisone (0.2 micrograms/ml) did not suppress basal ACTH secretion during 30 min but significantly (P less than 0.05) inhibited stimulation produced by rat stalk median eminence extract. Arginine vasopressin (dose range, 1-9 ng/ml) significantly (P less than 0.025) stimulated both ACTH and LPH secretion during 30 min. The patterns of response were again very similar. Serotonin (dose range, 0.01-10 micrograms/ml) did not affect ACTH secretion during incubations of 30 min to 4 h. The results obtained with the cell cultures of a human corticotropic cell adenoma concur with in vivo findings of incomplete autonomy of secretion, parallel secretion of ACTH and LPH in response to provocative stimuli, and suppression by corticosteroids. The technique has potential for exploring the cellular mechanisms controlling secretion by human corticotropic adenomas as well as the nature of the hormones produced.

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.

Chromatographic characterization of adsrenocorticotrophin in human plasma.

A chromatographic procedure has been developed for the characterization of ACTH- and lipotrophin- (LPH) related peptides in human plasma under acid-dissociating conditions to minimize artifacts of protein binding. The recovery and sensitivity of this method permits identification of ACTH at normal physiological levels in the circulation. Plasma profiles obtained from normal subjects and patients with pituitary dependent Cushing's disease, Addison's disease and Nelson's syndsrome showed only one significant peak of ACTH activity eluting in the position of purified native human 1--39 ACTH. However, the plasma profiles obtained from all the patients with the ectopic ACTH syndrome demonstrated a second peak of immunoreactive larger-molecular-weight ACTH and in some plasma samples this was the only form of ACTH observed. This larger-molecular-weight ACTH eluted midway between the void volume and 1--39 ACTH and co-eluted with a protein marker of molecular weight 22 000.

beta-Endorphin and beta-MSH in human plasma.

The aim of this study was to establish whether or not a peptide with chromatographic and immunological properties of beta-endorphin exists in human plasma. Using direct chromatography under conditions designed to minimize generation of beta-endorphin and beta-MSH from beta-LPH, we invariably found a peptide with beta-endorphin immunoreactivity eluting in the position of beta h-endorphin on gel chromatography in samples of plasma from patients with elevated ACTH and LPH levels. beta-MSH was only found in the plasma of one patient with the ectopic ACTH syndrome.