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Growth hormone-releasing factor (GRF) is believed to mediate both neural and hormonal control of the release and perhaps synthesis of growth hormone (GH) from the anterior pituitary. Its presence in only minute quantities in the hypothalamus hampered for many years efforts to determine its structure. Recently, two groups independently characterized a peptide from two human pancreatic tumours that possessed GH-releasing activity. Antibodies to this peptide have revealed immunoreactive material in the hypothalamus of several primate species. Furthermore, an apparently identical peptide has now been isolated directly from human hypothalamus. Using both oligonucleotide and antibody screening of a cDNA expression library, we have isolated a recombinant clone encoding human pancreatic GRF (hpGRF) within a larger precursor protein. The nucleotide sequence predicts that processing of GRF from the precursor should generate two additional peptides of unknown function. Restriction analysis of genomic DNA indicates that there is probably a single human GRF gene and suggests that the pancreatic tumour and hypothalamic proteins are encoded by an identical mRNA.

Alternative processing of the RNA transcribed from the calcitonin gene appears to result in the production of a messenger RNA in neural tissue distinct from that in thyroidal 'C' cells. The thyroid mRNA encodes a precursor to the hormone calcitonin whereas that in neural tissues generates a novel neuropeptide, referred to as calcitonin gene-related peptide (CGRP). The distribution of CGRP-producing cells and pathways in the brain and other tissues suggests functions for the peptide in nociception, ingestive behaviour and modulation of the autonomic and endocrine systems. The approach described here permits the application of recombinant DNA technology to analyses of complex neurobiological systems in the absence of prior structural or biological information.

Inhibin (I) a gonadal hormone glycoprotein which suppresses follicle-stimulating hormone (FSH) secretion from the anterior pituitary, is a heterodimer consisting of an alpha subunit and one of two distinct beta subunits. S1 nuclease analysis has revealed that RNAs encoding all three subunits (alpha, beta A and beta B) are expressed in rat brain. We report here on the localization, and a potential function, of inhibin beta in the rat brain. A cell group centred in the nucleus of the solitary tract (NTS), a major recipient of visceral sensory information, was stained immunohistochemically with antisera against synthetic fragments of I beta, but not I alpha. The distribution of I beta-stained fibres is consistent with known NTS projections, and includes a prominent projection to oxytocinergic aspects of the magnocellular neurosecretory system.

The importance of the hypothalamus in the regulation of growth hormone (GH) secretion from the adenohypophysis has been supported by experimental and clinical evidence. It has been observed that GH secretion is in part controlled by hypothalamic GH release-inhibiting factors, two forms of which, somatostatin-14 (ref. 5) and somatostatin-28 (refs 6-9), have been characterized. Peptides with GH-releasing activity were recently purified from two human pancreatic tumours of acromegalic patients and characterized. Synthetic versions of these human pancreatic tumour GH-releasing factors (hpGRFs) were found to be potent GH secretagogues in vitro and in vivo and to exhibit actions and interactions similar to those observed with partially purified hypothalamic GRF. GH-releasing peptides have been purified from hypothalami of various species but the structure of natural hypothalamic GRF has not yet been reported. We now describe the purification, sequence analysis and synthesis of a 43-residue polypeptide isolated from rat hypothalamic extracts on the basis of its ability to stimulate GH secretion from cultured rat adenohypophyseal cells. The native polypeptide and its synthetic replicate do not differ significantly in their biological potencies and intrinsic activities. We propose that this polypeptide, which shows 67% homology with the corresponding N-terminal 43 residues in hpGRF-44, is the long-sought rat hypothalamic GRF (rhGRF).

Corticotropin-releasing factor (CRF), the recently characterized and synthesized 41-amino acid polypeptide isolated from ovine hypothalami, has been shown to be a potent stimulator of adenohypophyseal beta-endorphin and corticotropin (ACTH) secretion both in vitro and in vivo. In common with other regulatory peptides, CRF has also been demonstrated to possess extra-hypophysiotropic roles. Indeed, intracerebroventricularly (i.c.v.) administered CRF elicits several endocrine and behavioural responses compatible with the concept that this peptide could be a key signal in coordinating the organism's endocrine and behavioural responses to stressful and other adaptive stimuli. We now provide the first evidence for neurally placed CRF in the control of a specific hormone-dependent behavioural response and unequivocally demonstrate an extremely potent suppressive effect of CRF on sexual behaviour in the female rat when microinfused into the arcuate-ventromedial area of the hypothalamus (ARC-VMH) and the mesencephalic central grey (MCG).

The hypothalamic-pituitary-adrenocortical axis is activated in pregnancy and parturition. Levels of immunoreactive corticotrophin releasing factor (irCRF), immunoreactive adrenocorticotropic hormone (irACTH) and cortisol concentrations in maternal plasma are elevated throughout gestation, increase further during labour and fall precipitously after parturition. The placenta contains biologically active CRF and ACTH and it has been suggested that the placenta produces these peptides during pregnancy. Here we show that irCRF is located in the cytotrophoblast cells of placenta collected at term. Using a monolayer primary culture of human placental cells we have found that CRF stimulates secretion of peptides containing the ACTH sequence in the placenta in a dose-dependent manner, as it does in the pituitary. This effect is reversed by a CRF antagonist and is mimicked by dibutyryl cyclic AMP and forskolin. Glucocorticoids, which suppress the secretion of pituitary ACTH, were found to have no influence on release of irACTH by the placenta. Oxytocin and prostaglandins stimulate irACTH and irCRF secretion from cultured placental cells and the irACTH-releasing activity of two prostaglandins is partially reversed by a CRF antagonist. Thus CRF may be involved in the paracrine regulation of placental irACTH secretion.

A variety of hypophysiotropic peptides or proteins have been reported to be present in mammalian gonads. Inhibin, a hormone that under most circumstances selectively suppresses the secretion of follicle-stimulating hormone (FSH) but not luteinizing hormone (LH), has been isolated from the gonadal fluids of several species and characterized as a heterodimeric protein consisting of alpha- and beta-polypeptides associated by disulphide bonds. The complete amino-acid sequences of the precursors of porcine and human inhibin alpha-subunits and two distinct porcine inhibin beta-subunits (beta A and beta B) have been deduced from complementary DNA sequences. Gonadotropin releasing peptides have also been found in the gonad and have generally been shown to be active in radioreceptor assays for gonadotropin releasing hormone (GnRH) but to exhibit different chromatographic and immunological characteristics from those of GnRH. During our purification of inhibin from porcine follicular fluid, we noted fractions that could stimulate the secretion of FSH by cultured anterior pituitary cells. We report here the purification of an FSH releasing protein (FRP) and its characterization by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions and by partial sequence analysis. Our results indicate that porcine gonadal FRP is a homodimer consisting of two inhibin beta A-chains linked by disulphide bonds. FRP is highly potent (50% effective concentration (EC50) approximately 25 pM) in stimulating the secretion and biosynthesis of FSH but not of LH or any other pituitary hormone. In contrast to the effects of GnRH and other reported gonadal gonadotropin releasing fractions, the action of FRP is not mediated by GnRH receptors.

Hypothalamic growth hormone-releasing factors (GRFs) have been purified recently from human pancreatic (hp) tumours and from rat hypothalamus (rh). GRF peptides have strong homology with peptides of the glucagon, vasoactive intestinal polypeptide and PHI-27 family. Aside from their potent actions on release of somatotropin, no other biological actions of GRFs have been reported. GRF has been localized in neurones bordering the ventromedial hypothalamic nucleus, a region associated frequently with experimental analysis of feeding behaviour. We now report that intracerebroventricularly (i.c.v.)-administered rhGRF and hpGRF(1-40) in doses of 0.2, 2.0 and 20.0 pmol, produced an increase in food intake in hungry rats. This effect seemed to be specific to GRF as i.c.v. injections of a structurally related but physiologically inactive peptide in the same doses had no effect on feeding. In addition, peripheral injections of rhGRF or growth hormone had no effect on food intake, suggesting that the present effects may be mediated centrally. Injections (i.c.v.) of rhGRF (0.2, 2.0 and 20.0 pmol) had no effect on general activity, suggesting that GRF does not produce nonspecific arousal.

Growth hormone releasing factors (GRFs) have been isolated from human pancreatic tumours (hGRF) and rat hypothalamus (rhGRF). The response to GRF at the pituitary level can be modulated by other factors, including glucocorticoids, thyroid hormones, somatostatin and other neuropeptides and somatomedins. Glucocorticoids enhance GRF-induced growth hormone (GH) secretion in primary cultures of rat anterior pituitary cells, and the synthetic glucocorticoid dexamethasone has recently been shown to increase the amounts of GH released in freely moving rats in response to submaximal doses of intravenous GRF. To investigate whether somatotroph sensitivity to GRF is modulated at its receptor level, we have developed a radioreceptor assay using an iodinated analogue of hGRF as radioligand. We report here that the relative binding affinities of rGRF, hGRF and the two analogues are correlated with their in vitro biological potencies. Further, the number of GRF binding sites is drastically decreased in cells deprived of glucocorticoids either in vivo or in vitro.

Inhibin is a hypophysiotropic hormone which selectively suppresses the secretion of pituitary follicle-stimulating hormone. It has been isolated from gonadal fluids and characterized as a protein heterodimer consisting of an alpha subunit and one of two beta subunits (beta A or beta B). FSH-releasing protein (FRP), also named activin, is a dimer consisting of two inhibin beta-chains. A factor from conditioned medium of a leukaemia cell line has been isolated which can induce mouse Friend cells to become benzidine-positive, and which shares a similar N-terminal sequence with porcine FRP. In this report, we find that FRP and inhibin modulate both the induction of haemoglobin accumulation in a human erythroleukaemic cell line, K562, and the proliferation of erythroid progenitor cells in human bone marrow culture. These two proteins could constitute a novel humoral regulatory control of erythropoiesis which would involve two types of related protein dimers with functionally opposite effects.