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Corticotrophin-releasing factor in the human placenta
When the occurrence in the human placenta of significant amounts of the 41 residue hypothalamic peptide, corticotrophin-releasing factor (CRF), was first reported in the literature (Shibasaki et al. 1982), many endocrinologists engaged in research on the hypothalamic-pituitary-adrenal (HPA) axis were somewhat sceptical, particularly about the high concentrations of immunoreactivity that were being found. We had come to accept that the placenta produced a number of protein hormones, in particular, homologues of the pituitary protein and glycoprotein hormones, namely placental lactogen and chorionic gonadotrophin, which are secreted into the maternal bloodstream in quite significant amounts. But, invariably the concentrations in placental tissue of the smaller peptide hormones, both of the pituitary and hypothalamic type, were some orders of magnitude less (Kreiger 1982). The initial report (Sasaki et al. 1984) of CRF in peripheral blood of pregnant women in their third trimester at concentrations normally associated
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Available bioassays for corticotrophin releasing factor (CRF) (McCann & Haberland, 1959; De Wied, 1961; Arimura, Saito & Schally, 1967; Hiroshige, Kunita, Yoshimura & Itoh, 1968) are unsuitable for routine purposes because of their technical complexity and lack of sensitivity. Assays employing hemipituitaries in vitro (Saffran & Schally, 1955; Sadow, Penn & Knight, 1972) suffer from the inability of substances to diffuse freely into and out of those cells which are shielded from the bathing medium. The use of isolated pituitary cells overcomes this disadvantage and the batch-type incubation of cells has been described by Portanova, Smith & Sayers (1970), Portanova (1972) and Portanova & Sayers (1973). Corticotrophs, however, contain large stores of corticotrophin (ACTH) and consequently their lysis and death during incubation can lead to high background values.
The recently developed technique of cell-column perfusion (Lowry & McMartin, 1974) for the measurement of releasing factor activity offers the advantage that
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The corticotrophin releasing (CRF) activity of stalk median eminence (SME) extracts from homozygous Brattleboro (DIhomo) rats was significantly less than that of the heterozygous (DIhet) rats which, in turn, was significantly less than normal (P < 0·01). The bio- and immunoactivities of LH-releasing hormone (LH-RH) were not significantly different. No detectable immunoactive vasopressin was found in DIhomo SME and the vasopressin content of DIhet SME was less than normal. Chromatography of an extract of SME from 20 DIhomo rats on BioGel P2 resulted in a loss of CRF activity and the emergence of two regions of CRF activity: the peak at the void volume of the column and the later eluting peak which also had some LH-RH bioactivity but no immunoactivity. The third and major CRF peak found in normal SME, which co-elutes with vasopressin, was absent from the DIhomo chromatogram. The DIhomo LH-RH chromatogram was normal. When synthetic arginine-vasopressin was added to Brattleboro SME in amounts equivalent to those found in normal SME the CRF bioactivity was dramatically potentiated. It was concluded that Brattleboro rats have a specific defect for CRF at the hypothalamic level and this appears to be genetically linked to the synthesis of vasopressin which in itself is also essential for the stability and full expression of CRF bioactivity.
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Intermediate and anterior lobes from the pituitary glands of female Wistar rats were freshly dissected and chromatographed on Sephedex G-50 and BioGel P6. Fractions were monitored with radioimmunoassays for NH2- and CO2H-terminal adrenocorticotrophin (ACTH), α-melanocyte-stimulating hormone (α-MSH), and α- and β-endorphin. A large molecular weight, glycosylated form of corticotrophin-like intermediate lobe peptide (CLIP) which we have termed big CLIP, and a β-MSH-like molecule were identified in the pars intermedia and in both lobes, a major peak of activity with the elution characteristics and cross-reactivity of γ-lipotrophic hormone (γ-LPH) was detected. In the pars distalis, the larger peptides 1–39 ACTH and β-LPH predominated, whereas in the pars intermedia, the smaller peptides α-MSH, CLIP, β- and α-endorphin were more abundant. Chromatography of rat plasma revealed peaks of immunoreactivity in the corresponding positions to those detected in the pituitary gland.
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Combined rostral, median and neurointermediate lobes from 650 pituitary glands of the dogfish Squalus acanthias were extracted in 0·1 m-HCl and subjected to filtration on Sephadex G-50. Fractions were monitored for ACTH, α-MSH, γ-MSH and endorphin by heterologous radioimmunoassay, corticotrophin-like intermediate lobe peptide (CLIP) and γ-MSH by homologous radioimmunoassay, and methionine enkephalin after enzymatic digestion. The majority (about 99%) of the immunoreactivity detected was present as small peptides, with α-MSH, CLIP and γ-MSH predominant. The single peaks of ACTH, α-MSH and CLIP contrasted with many distinct peaks of endorphin-like immunoreactivity. The γ-MSH radioimmunoassays monitored different peptides; the heterologous assay revealed a single major peak, while the homologous assay detected a number of distinct small peptides. The results suggested that there may be more than three distinct forms of MSH in the dogfish pituitary gland. Small amounts of much larger proteins were detected by a number of the radioimmunoassays, suggesting that peptides related to ACTH, γ-MSH and lipotrophin are derived from common pro-opiocortin-type precursor molecules in this phylogenetically ancient cartilaginous fish.
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SUMMARY
A reproducible method for isolating intact cells from the adrenal glands of a small number of rats (four to six) is described. The incubation conditions and fluorometric corticosteroid assay were modified to enable a large number of incubations to be carried out with each batch of cells. Linear regression analysis of log-dose—response curves for adrenocorticotrophin (ACTH) analogues yields potency ratios with 95% confidence limits which are usually less than ± 20%. The ED50 of the assay is about 50 pg/ml for natural ACTH and 7 pg/ml for corticotrophin-(1–24)-tetracosapeptide. Insulin and prostaglandin E2 had no effect on the steroidogenic response to ACTH while cyclic AMP at high doses was steroidogenic but gave a much steeper log-dose—response curve than ACTH. α- and β-melanocytestimulating hormone (MSH) were both agonists but only at concentrations 1 × 106 times higher than those of ACTH. Cells isolated from rabbit adrenals were similar to those from the rat in their responsiveness to ACTH but were unresponsive to amounts of MSH sufficient to stimulate rat adrenal cells.
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SUMMARY
At least seven radioactive peptides, which fractionated on Biogel P6, were found in rat neurointermediate lobes after incubation for 6 h with [14C]proline. Only three of these could be tentatively identified; one as α-melanocyte-stimulating hormone (α-MSH) and two as forms of corticotrophin-like intermediate lobe peptide (CLIP). One other crossreacted partially with a β-melanocyte-stimulating hormone (β-MSH) antiserum, was acidically charged and eluted on Biogel P6 in roughly the same position as ACTH. The other three peptides showed no resemblance to α-MSH, CLIP, β-MSH or ACTH.
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ABSTRACT
We report the purification of human corticotrophin-releasing factor-binding protein (hCRF-BP) using repeated affinity chromatography (with the aid of synthetic CRF immobilized on Sepharose 4B solid phase) followed by gel filtration. Presence of the binding protein was tracked throughout the procedure by its ability to inhibit binding of 125I-labelled hCRF to an hCRF antiserum; normal human plasma exhibits 80% inhibition in this system whereas sheep plasma, which does not contain an hCRF-BP, has no effect. Affinity cross-linking of 125I-labelled hCRF to the purified hCRF-BP was performed using disuccinimidyl suberate (1 mmol/l). SDS electrophoresis of the purified CRF cross-linked binding protein followed by radioautography resulted in one major band of M r 37 000 which corresponded to our original molecular weight estimate based on the elution position of the binding protein on Sephacryl S-200. A 107-fold purification of the hCRF-BP resulted in a preparation estimated to be 95% pure, with an overall yield of 5%.
Journal of Endocrinology (1989) 122, 23–31
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SUMMARY
Pig posterior pituitary lobe powder contains a peptide structurally identical to the 18–39 portion of porcine adrenocorticotrophin (ACTH). Its extraction by several different procedures is described and its susceptibility to degradation in the presence of 5% acetic acid has been noted. This degradation has been ascribed to the presence of acid proteases in the acetone-dried powder of the posterior pituitary lobe. The peptide has been isolated by chromatography on Biogel P6 and DEAE-cellulose, and characterized by enzyme fragmentation studies. It resembles the peptide isolated from rat neurointermediate lobes termed 'corticotrophin-like intermediate lobe peptide' and it is suggested that it is derived by the intracellular cleavage of ACTH in the pars intermedia cells, with subsequent formation of α-melanocyte-stimulating hormone from the other adrenocorticotrophic fragment.
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SUMMARY
The pars intermedia of the rat pituitary contains a peptide resembling the 18–39 portion of adrenocorticotrophic hormone (ACTH), which has been termed 'corticotrophin-like intermediate lobe peptide' (CLIP). It can be detected by its cross-reaction with an antiserum directed against the CO2H-terminal portion of the ACTH molecule; it has an amino acid composition identical to the 18–39 portion of human ACTH, except for one less glycine and an extra valine residue, and it is rapidly released from neurointermediate lobes maintained in organ culture. The pars intermedia also contains a peptide with an amino acid composition and biological potency identical to that of melanocyte-stimulating hormone (α-MSH) isolated from other mammals, and which accounts for the bulk of melanocyte-stimulating activity in the pituitary. Rat ACTH resembles human ACTH in amino acid composition, except for an extra valine and one less glycine residue. On the basis of these data it is proposed that ACTH is the precursor of α-MSH and CLIP, which are both present in the cells of the pars intermedia.
