Joachim Spiess

Chemical and biological characterization of the inhibin family of protein hormones.

Publisher Summary This chapter discusses the chemical and biological characterization of the inhibin family of protein hormones, which is a family of peptides isolated from the follicular fluid or rete testis fluid on the basis of their ability to inhibit the secretion of the follicle-stimulating hormone (FSH) by cultured rat anterior pituitary cells. It also reviews the possible roles of inhibin and fibre-reinforced plastic (FRP)/activin in placenta, brain, and bone marrow. Inhibin-related dimers are broadly distributed anatomically and have powerful activities in several biological systems where inhibin and FRP/activin often exhibit opposite effects. While the physiologic roles of inhibin to regulate FSH secretion in the female rat and immature male rat are strongly supported, the significance of these hormones within the gonad, brain, placenta, and bone marrow have yet to be placed in in vivo context. Although the panoply of functions of inhibin and FRP/activin are certainly incompletely understood at this time, this family has already demonstrated a powerful mechanism for the generation of signal diversity whereby differential subunit association can result in the generation of dimers with opposing biological actions in multiple tissues.

Characterization of rat hypothalamic growth hormone-releasing factor.

The importance of the hypothalamus in the regulation of growth hormone (GH) secretion from the adenohypophysis has been supported by experimental and clinical evidence1–3. It has been observed that GH secretion is in part controlled by hypothalamic GH release-inhibiting factors4, 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 characterized10–14. Synthetic versions of these human pancreatic tumour GH-releasing factors (hpGRFs) were found to be potent GH secretagogues in vitro11,12,15 and in vivo12,16,17 and to exhibit actions and interactions similar to those observed with partially purified hypothalamic GRF12. GH-releasing peptides have been purified from hypothalami of various species4,15,18,36 but the structure of natural hypothalamic GRF has not yet been reported19. 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).

Purification and partial characterization of inhibin from porcine follicular fluid.

Inhibin, a protein of gonadal origin that suppresses the basal secretion of follicle stimulating hormone by anterior pituitary cells has been purified from porcine follicular fluid. Using several RP-HPLC steps and gel filtration under denaturing conditions, we obtained a fraction approximately ten thousand fold purified which showed one band on SDS PAGE and in the same experiment two bands after reduction (MW ca 14K and ca 18K) suggesting a molecular weight of 32K for inhibin. Edman degradation of isolated inhibin and carboxymethylated chain A indicated that the first 6 residues were H-Ser-Thr-Ala-Pro-Leu-Pro-; by subtraction, the first 3 residues of chain B could be deduced to be H-Gly-Leu-Glu-. EC50 was ca 0.3 ng/ml or 10 pM in our in vitro pituitary cell culture assay. Antibodies to residues 1-6 were raised which could immunoneutralize purified inhibin activity in an in vitro assay.

Corticotropin-releasing factor: Effects on the sympathetic nervous system and oxygen consumption

Corticotropin-releasing factor administered intracerebroventricularly produces prolonged elevation of plasma concentration of epinephrine, norepinephrine and glucose. These hormonal changes are associated with an increase in motor activity and oxygen consumption. No change in body temperature is observed. CRF produces changes in animal physiology that are similar to those observed in response to stress.

EFFECTS OF HUMAN PANCREATIC TUMOUR GROWTH HORMONE RELEASING FACTOR ON GROWTH HORMONE AND SOMATOMEDIN C LEVELS IN PATIENTS WITH IDIOPATHIC GROWTH HORMONE DEFICIENCY

Human pancreatic tumour growth hormone releasing factor (hpGRF-40) 10 micrograms/kg was administered intravenously to 6 normal young men and 12 adult patients who had presented in childhood with growth hormone (GH) deficiency (7 patients had isolated GH deficiency, 4 had multiple anterior pituitary hormone deficiencies, and 1 had Hand-Schüller-Christian [HSC] disease). hpGRF-40 administration increased serum GH concentrations in all normal subjects and in 3 of 7 patients with isolated GH deficiency and in the 1 with HSC disease; however, the mean serum GH concentration in the patients who responded was less than that of the normal subjects. Somatomedin C concentrations were increased 24 h after a single dose of hpGRF-40 in 8 of 10 patients with GH deficiency. All subjects experienced flushing in response to hpGRF-40. A patient with isolated GH deficiency received 0.33 micrograms/kg hpGRF-40 every 3 h for 5 days. Despite the modest increase in GH in response to a subsequent dose of 10 micrograms/kg hpGRF-40, serum somatomedin C levels increased within 12 h from 0.06 to 0.1 U/ml and peaked at 0.36 U/ml at 72 h; in addition the patient with HSC disease, treated with hpGRF-40 daily for 5 days, demonstrated an increase in somatomedin C from 0.4 to 0.58 U/ml. The increase after hpGRF-40 in serum GH levels in this patient and the similar or greater responses in 3 of 7 patients suggest that at least some of these patients may have hypothalamic GH-releasing-hormone deficiency. hpGRF-40 may be useful in distinguishing pituitary disease from hypothalamic disease. After hpGRF-40 administration serum somatomedin C levels may increase without a change in serum immunoreactive GH concentrations. Further studies are needed to determine whether hpGRF-40 is useful in promoting linear growth in children with GH deficiency.

Comparison of the biologic actions of corticotropin-releasing factor and sauvagine

Corticotropin-releasing factor (CRF), a peptide isolated from ovine hypothalamus, and sauvagine, a peptide isolated from frog skin, share significant structural homology and elicit a number of similar biological responses. CRF is more potent than sauvagine in stimulating pituitary ACTH secretion. Sauvagine, however, is 5-10 times more potent than CRF to act within the brain to increase plasma levels of catecholamines and glucose and to elevate mean arterial pressure. Sauvagine is likewise more potent than CRF to act outside the brain to increase superior mesenteric artery flow and plasma glucose concentrations and to decrease mean arterial pressure. CRF and sauvagine produce important effects representative of biologically active peptides.

Mechanism of action of somatostatin: An overview of receptor function and studies of the molecular characterization and purification of somatostatin receptor proteins☆

To determine whether somatostatin receptor subtypes arise from molecular heterogeneity of the receptor protein, we have cross-linked the putative receptor in normal rat tissues and in AtT-20 and GH3 cells, both chemically with SS-14, SS-28 and Tyr3 SMS ligands, as well as by photoaffinity labeling with an azido derivative of Tyr3 SMS (EE 581). Three prominent somatostatin receptor proteins of 58-kDa, 32-kDa, and 27-kDa size have been identified. These proteins exhibit a tissue-specific distribution, ligand selectivity, and relative preference for SS-14 and SS-28 binding, and thus qualify as somatostatin receptor subtypes. Using EE 581 as a photoaffinity probe, the 58-kDa and 32-kDa proteins have been purified to homogeneity from brain and AtT-20 cells by successive SDS-PAGE. The 58-kDa form has been trypsinized and amino acid sequence data obtained from four tryptic fragments. With the help of synthetic oligonucleotides derived from these sequences, work is currently in progress to clone the 58-kDa protein to elucidate its complete sequence, its expression, and its functional relationship to the somatostatin receptor and its pharmacological subtypes.

Isolation and Characterization of Hypothalamic Growth-Hormone Releasing Factor from Common Carp, Cyprinus carpio

A growth hormone-releasing factor (GRF)-like peptide was isolated from the hypothalamus of common carp, Cyprinus carpio, by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against rat GRF, and multiple steps of HPLC using octadecyl columns. Based on Edman degradation and peptide mapping, this teleost GRF was established to be a 45-residue peptide with the following primary structure: His-Ala-Asp-Gly-Met-Phe-Asn-Lys-Ala-Tyr-Arg-Lys-Ala-Leu-Gly-Gln-Leu-Ser- Ala-Arg - Lys-Tyr-Leu-His-Thr-Leu-Met-Ala-Lys-Arg-Val-Gly-Gly-Gly-Ser-Met-Ile-Glu- Asp-Asp-Asn-Glu-Pro-Leu-Ser. Carp GRF is closely related structurally to peptides of the glucagon-secretin superfamily, and more particularly to mammalian vasoactive intestinal peptide (VIP) precursors and the N-terminal portion of mammalian GRFs. A synthetic replicate of this peptide is highly potent [50% effective dose (ED50) approximately 0.08 nM] in stimulating GH release from cultured goldfish pituitary glands and in elevating serum GH levels 30 min after injection (0.1 micrograms/g) in goldfish.

Investigation of larger forms of somatostatin in pigeon pancreas and rat brain.

Abstract Either ribosomal or nonribosomal mechanisms1 could be considered for the biosynthesis of the polypeptide somatostatin. Since it seems improbable on the basis of investigations of Blobel et al.2,3 that small secretory peptides such as somatostatin are directly synthesized on ribosomes, a ribosomal mechanism of somatostatin biosynthesis would require a larger precursor molecule which could be synthesized on the ribosomes and then be enzymatically processed to generate the tetradecapeptide somatostatin. The possibility that hypothalamic extracts might contain larger somatostatin species was proposed by Schally et al.4 and in a report from our laboratories.5 Arimura et al.6 found two immunoreactive somatostatin species in the extracts from the stomach and pancreas of the rat. One species eluted in Sephadex G-25 gel chromatography with the tetradecapeptide somatostatin. Most of the pancreatic somatostatinlike activity (SLA) was eluted in the void volume, possibly representing a larger SLA species. Recent investigations of Dupont and Alvarado-Urbina,7 however, raised doubts about the existence of a big pancreatic somatostatin. According to this report, the big pancreatic somatostatin species can be converted into the small form, which is eluted with the tetradecapeptide somatostatin in gel chromatography, by treatment with 8 M urea independent of the presence of thioglycol. In this paper, we report our investigations of larger somatostatin species using extraction and purification procedures that minimize protein-protein interactions and protease activities.

High-performance liquid chromatographic purification of peptide hormones: Ovine hypothalamic amunine (corticotropin releasing factor)☆

Abstract Amunine, or corticotropin releasing factor (CRF), a 41-peptide amide, has been isolated from ovine hypothalami. Tissues were extracted, defatted, and partitioned prior to being sized by gel permeation. The active zone followed by a sensitive in vitro assay (CRF-mediated corticotropin release from pituitary cells in monolayer cultures quantitated by a specific corticotropin radioim munoassay) could be purified by reverse-phase high-performance liquid chromatography. Column characteristics (i.e., properties of the derivatized silicas including pore size), selection of mobile phases, and temperature effects are discussed.