Sally J. Ratter

STUDIES ON CIRCULATING MET-ENKEPHALIN AND β-ENDORPHIN: NORMAL SUBJECTS AND PATIENTS WITH RENAL AND ADRENAL DISEASE

Studies were performed to define the responses of plasma met‐enkephalin to various endocrine and pathological stimuli and to determine the relationship between plasma β‐endorphin and met‐enkephalin. During insulin‐induced hypoglycaemia ACTH, β‐LPH and β‐endorphin immunoreactivity rose in parallel, but plasma met‐enkephalin did not change significantly. Sephadex G75 chromatography of samples taken at the time of the peak response (45 min) confirmed the rise in both β‐LPH and β‐endorphin. During administration of dexamethasone, 0·5mg 6 hourly for 48 h, plasma cortisol and ACTH became undetectable at 24 h, and β‐LPH and β‐endorphin fell significantly by 24 h and were undetectable by 48 h; plasma met‐enkephalin, however, showed no significant change. Nine adrenalectomized patients with Cushings disease and four patients with Addisons disease had elevated plasma ACTH, β‐LPH and β‐endorphin but normal plasma met‐enkephalin levels. Each of ten patients with renal failure had markedly elevated plasma met‐enkephalin immunoreactivity which co‐eluted with synthetic met‐enkephalin on BioGel P4 chromatography. Trypsin and carboxypeptidase‐B digestion of the P4 chromatography fractions generated met‐enkephalin immunoreactivity in earlier fractions, indicating the presence of a potential high molecular weight met‐enkephalin precursor.

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

Basal and stimulated secretion of N‐terminal pro‐opiocortin (Pro‐γ‐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‐chro‐matographic 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.

DEVELOPMENT OF A RADIOIMMUNOASSAY FOR AN AMINO‐TERMINAL PEPTIDE OF PRO‐OPIOCORTIN CONTAINING THE γ‐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 β‐LPH, using an antiserum which recognizes residues near the γ‐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.

Endogenous opioid peptides in pregnancy.

Summary. Plasma levels of two endogenous opioid peptides, β‐endorphin and met‐enkephalin, as well as immunoreactive N‐terminal β‐lipotrophin (N‐LPH) were studied in normal pregnant women from 8 to 41 weeks gestation. A total of 116 samples were assayed for β‐endorphin‐like immunoreactivity (C‐LPH), 103 for N‐LPH and 75 for met‐enkephalin. Plasma β‐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 adreno‐corticotrophin (ACTH)/β‐LPH and β‐endorphin secretion throughout pregnancy, while the unchanged met‐enkephalin levels are compatible with its known derivation from a separate precursor system.

SECRETION OF ACTH, LPH AND β‐ENDORPHIN FROM HUMAN PITUITARY TUMOURS IN VITRO

Basal and stimulated secretion of immunoreactive ACTH, LPH and β‐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 tumour cells released immunoreactive peptides with the elution profiles of αh (1–39) ACTH, βh‐LPH, γh‐LPH and βh‐endorphin confirming that βh‐endorphin is secreted by human pituitary tumour cells and is not formed by enzymic cleavage from βh‐LPH in blood. No α‐ or βh‐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, β‐LPH, γ‐LPH and γ‐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 Cushings disease and Nelsons syndrome can be studied in vitro and that such studies may contribute to a greater understanding of the aetiology of these diseases.

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.

β-ENDORPHIN AND β-MSH IN HUMAN PLASMA

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

A CASE OF PITUITARY DEPENDENT CUSHING'S DISEASE WITH CLINICAL AND BIOCHEMICAL FEATURES OF THE ECTOPIC ACTH SYNDROME

A case of atypical pituitary dependent Cushings 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 patients Cushings 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.

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 Cushings 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.

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.