Assay of corticotropin releasing factor.
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Adrenocorticotropic Hormone↗
Biomedical subjects
W Vale
Publications and source records attributed to W Vale.
Effects of gonadotropin releasing hormone agonists and antagonists on reproductive functions.
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Animals↗
Characterization of rat hypothalamic corticotropin-releasing factor.
A polypeptide was purified from rat hypothalamic extracts on the basis of its high intrinsic activity to release corticotropin (ACTH) from cultured rat anterior pituitary cells and its immunoactivity in a radioimmunoassay directed against the NH2 terminus (residues 4-20) of ovine hypothalamic corticotropin-releasing factor (CRF). Based on Edman degradation, peptide mapping, and amino acid analysis, the primary structure of this rat CRF was established to be: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. The hypophysiotropic potency of synthetic rat CRF did not deviate significantly from the potencies of the isolated native peptide or of synthetic ovine CRF. The close structural relationship between rat and ovine hypothalamic CRF is indicated by an 83% sequence homology.
Amino Acid Sequence↗
Structural homology of corticotropin-releasing factor, sauvagine, and urotensin I: circular dichroism and prediction studies.
Three recently isolated peptides, whose sequences have been determined--the corticotropin (adrenocorticotropic hormone)-releasing factor of ovine origin, sauvagine, from the skin of the frog Phyllomedusa sauvagei, and urotensin I from the teleost fish, Catostomus commersoni--show high (greater than 50%) sequence homology. CD spectra of the three peptides in trifluoroethanol indicate predominantly helical character for these peptides. Analysis of the secondary structures by the Chou-Fasman method predicts that the overall structural organization of the peptides is the same. All three possess a long internal helix, spanning about 25 residues, connected by a turn region to a COOH-terminal structural element that is an alpha-helix in corticotropin-releasing factor and urotensin I and a beta-sheet in sauvagine. The values for helical content estimated from the prediction method agree reasonably well with those computed from the CD spectra. This agreement as well as the CD spectra of corticotropin-releasing factor fragment 5-33 support the specific assignments of helical regions derived from the Chou-Fasman analysis. The three peptides exhibit significantly less helical structure in water than in trifluoroethanol as indicated by CD spectra. Hydrophilicity profiles provided comparison of the three peptides in terms of their overall hydrophilicity and the location of the regions of maximal hydrophilicity. A unique distribution of hydrophilic and hydrophobic residues within the internal helices is revealed by helical wheel analysis. Patches of both types of residues are formed following a heptad (four/three) rule. Since the two patches are shifted by one residue relative to one another, together they occupy only one face of the helical surface, a feature distinct from other amphiphilic structures.
Amphibian Proteins↗
Surface properties of an amphiphilic peptide hormone and of its analog: corticotropin-releasing factor and sauvagine.
Synthetic corticotropin (adrenocorticotropic hormone)-releasing factor [CRF; for the sequence, see Vale, W., Spiess, J., Rivier, C. & Rivier, J. (1981) Science 213, 1394-1397] in aqueous solution exists predominantly as a random coil. At concentrations greater than 1 microM, the peptide shows a tendency to self-aggregate with a concurrent slight increase in the apparent alpha-helical content as measured by the CD spectrum. The alpha-helix formed by this molecule is highly amphiphilic--i.e., the hydrophilic and hydrophobic regions are segregated on opposite faces of the helix. As predicted from the potential amphiphilic structure, CRF binds avidly to the surface of single bilayer egg phosphatidylcholine vesicles. This binding appears to obey a simple Langmuir isotherm with the following parameters: Kd = 1.3 +/- 0.6 X 10(-7) M and capacity at saturation (N) = 11.0 +/- 1.0 mmol of peptide per mol of phospholipid. CRF also readily forms an insoluble monolayer at the air-water interface. The monolayer is composed of monomers of the hormone with molecular areas, A'0 = 22 A2 per amino acid, suggesting a compact secondary structure. Judged from the collapse pressure (19.0 +/- 0.1 dyne/cm; 1 dyne = 10 microN) of the monolayer, the amphiphilicity of CRF approximates that of plasma apolipoproteins, a class of proteins of the most pronounced amphiphilic character. These results suggest that the binding of CRF to the cell membrane is accompanied by the induction of an alpha-helical secondary structure and it is this predominantly helical form that is the biologically active form of the peptide.
Amino Acid Sequence↗
Characterization of a teleost gonadotropin-releasing hormone.
A peptide that is recognized by certain antibodies raised against mammalian gonadotropin-releasing hormone has been purified from extracts of salmon brains by gel filtration and high-performance liquid chromatography. The primary structure of this 10-residue peptide is less than Glu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2. This represents a difference of two amino acids between salmon and mammalian gonadotropin-releasing hormone and demonstrates that most of the molecule has been conserved during evolution. The synthetic form of salmon gonadotropin-releasing hormone is less potent than is mammalian gonadotropin-releasing hormone on mammalian cells and is biologically active in salmon.
Amino Acid Sequence↗
Comparison of the effect of several gonadotropin releasing hormone antagonists on luteinizing hormone secretion, receptor binding and ovulation.
The biological activity of three gonadotropin releasing hormone (GnRH) antagonists was evaluated in the following assays: suppression of GnRH-mediated luteinizing hormone (LH) secretion by cultured pituitary cells, suppression of the spontaneous LH release by ovariectomized rats, blockade of ovulation in regularly cycling females and inhibition of binding of a potent radiolabeled agonist to rat pituitary membrane homogenates. The peptides were: [Ac-delta 3Pro1,4FDPhe2, DTrp3,6]-GnRH (Antagonist 1); [Ac-delta 3Pro1,4FDPhe2,DNAL(2)3,6]-GnRH (Antagonist 2); and [Ac-DNAL(2)2,4FDPhe2,DTrp3,DArg6]-GnRH (Antagonist 3). All three antagonists exhibited similarly high potency in suppressing LH secretion in vitro, while Antagonist 1 was the most active peptide in the radioreceptor assay. When administered by gavage, Antagonist 3 exhibited the highest potency to inhibit LH secretion in gonadectomized rats and to block ovulation. Comparison of the oral versus the subcutaneous mode of administration of these analogs indicates that less than 1% is absorbed after gavage. However, these data demonstrate that the intragastric administration of GnRH antagonists can lower gonadotropin secretion and interfere with reproductive functions.
Administration, Oral↗
Influence of ethanol on reproductive functions of the adult male rat as a function of body weight.
Adult male rats were exposed to alcohol vapors for 3-4 weeks. Blood alcohol levels (BAL) ranged from 90 to 190 mg/100 ml. BAL of greater than or equal to 0-130 mg/100 ml were compatible with adequate body weight gains and were not associated with diminished plasma testosterone (T) levels or reduced sex organ weights. BAL greater than or equal to 180 mg/100 ml were associated with inhibition of androgen secretion only in those animals who failed to grow. From these results we conclude that in growing animals, the medium term exposure to ETOH leads to inhibition of reproductive parameters when high BALs induce weight loss, but not when adequate body growth is maintained.
Animals↗
Isolation, analysis of structure, synthesis, and biological actions of urotensin I neuropeptides.
The 41-residue neuropeptide urotensin I (UI), from the urophyses of two teleost fish species (Cyprinus carpio and Catostomus commersoni), was isolated and purified, and its amino acid sequence was determined and confirmed by synthesis of a fully active peptide. The UI peptide was found to be a close structural and biological homologue of the ovine hypothalamic corticotropin-releasing factor (CRF) and the frog skin peptide sauvagine; UI is, therefore, a phylogenetic prototype of this group of peptides. Extraction of urophyses in hot acetic or hydrochloric acid cleaves an amino terminal tripeptide yielding a fully active UI(4-41). The UI peptides are equipotent with the other two naturally occurring peptides (CRF and sauvagine) in the release of mammalian pituitary corticotropin (ACTH), but UI is several times more potent than the mammalian homologue in the stimulation of release of fish pituitary ACTH. The UI peptide and its mammalian or amphibian homologues have a long-lasting hypotensive action in mammals, via a uniquely selective vasodilatation in the superior (anterior) mesenteric vascular bed only. The significantly lower hypotensive vasodilatory action of the mammalian homologue (CRF) suggests a change in the unknown physiological role of the haemodynamic actions of the UI peptides in the mammalian gastrointestinal tract during phylogenetic progression from fishes to mammals.
Adrenocorticotropic Hormone↗
Facilitation of sexual receptivity in the female rat by a fragment of the LHRH decapeptide, Ac-LHRH.
The following fragments of LHRH were administered intraventricularly and tested for their ability to enhance sexual receptivity in ovariectomized, estrogen-primed female rats: LHRH1-6 NH2, Ac-LHRH5-10, des-Tyr5-LHRH and des-Gly10-LHRH. LHRH1-6 NH2 had no effect on sexual behavior compared to saline-infused controls. Ac-LHRH5-10, des-Tyr5 LHRH and des-Gly10-LHRH significantly elevated lordotic responding compared to saline-infused controls. The results suggest that the elements contained in the LHRH structure may be functionally differentiated and indicate that the active sequence for facilitation of mating behavior is contained in the last half of the LHRH molecule.
Animals↗
Corticotropin releasing factor (CRF) immunoreactivity in hypothalamic and extrahypothalamic nuclei of sheep brain.
In sheep, 26 brain areas of 62 investigated contained corticotropin releasing factor (CRF)-like immunoreactivity in concentrations higher than 0.5 ng CRF/mg protein. Most of the CRF in the brain was in the hypothalamus. The highest level of CRF in the sheep brain was in the median eminence, almost two orders of magnitude higher than anywhere else. All of the hypothalamic nuclei had measureable amounts of CRF. A number of extrahypothalamic regions such as amygdala, hippocampus, claustrum, cingulate cortex, habenula and certain lower brainstem nuclei had CRF but generally in very low concentrations.
Animals↗
Effect of synthetic ovine corticotropin-releasing factor. Dose response of plasma adrenocorticotropin and cortisol.
Synthetic ovine corticotropin-releasing factor (CRF) was administered to normal male volunteer subjects as an intravenous bolus or 30-s infusion. Doses of CRF ranging from 0.001 to 30 micrograms/kg body wt were administered, and plasma immunoreactive (IR)-ACTH and IR-cortisol concentrations were measured. The threshold dose appeared to be 0.01-0.03 micrograms/kg, the half-maximal dose 0.3-1 micrograms/kg, and the maximally effective dose 3-10 micrograms/kg. Basal concentrations of IR-ACTH and IR-cortisol were 14 +/- 7.6 pg/ml (mean +/- SD) and 5.6 +/- 2.2 micrograms/dl, respectively. IR-ACTH rose as early as 2 min after CRF injection, reached peak levels in 10-15 min, and declined slowly thereafter. IR-cortisol rose at 10 min or later and reached peak levels in 30-60 min. At a dose of 30 micrograms/kg, neither IR-ACTH nor IR-cortisol fell from peak levels of 82 +/- 21 pg/ml (mean +/- SE) and 23 +/- 1.4 micrograms/dl, respectively, during the 2-h course of the experiment, indicating that CRF has a sustained effect on ACTH release and/or a prolonged circulating plasma half-life. There was little or no increase in the levels of other anterior pituitary hormones. At doses of 1 microgram/kg and higher, facial flushing, tachycardia, and, in some subjects, a 15-29-mmHg decline in systemic arterial blood pressure were observed, even though blood volume was replaced and the subjects remained supine. These data indicate that synthetic ovine CRF is a very potent and specific ACTH secretagogue in man. Administered with caution until its vasomotor effects are more fully defined, CRF promises to be a safe and very useful investigative, diagnostic, and, possibly, therapeutic agent in man.
Adrenocorticotropic Hormone↗
Structure-activity relationships of somatostatin analogs in the rabbit ileum and the rat colon.
UNLABELLED: Somatostatin increases absorption of electrolytes and inhibits diarrhea in patients with endocrine tumors and short bowel syndrome. In an attempt to develop a gut-specific somatostatin analog, each amino acid in the somatostatin molecule was replaced with L-alanine, deleted, or substituted with its D-isomer. The potency of each analog to stimulate ion transport in the rabbit ileum was then determined using the modified Ussing chamber technique. The results were compared to the ability of each analog to inhibit the stimulated release of growth hormone from cultured rat anterior pituitary cells and to inhibit the arginine-stimulated release of insulin and glucagon in the rat in vivo. Analogs that showed gut selectivity were then tested for their ion transport properties in the rat colon. RESULTS: (a) Substitution with L-alanine or deletion of the amino acid at position 6, 7, 8, or 9 and deletion of Threonine(10)-produced analogs with significantly reduced ion transport properties to <4% of somatostatin's action. The substitution also markedly reduced the ability of the compounds to inhibit the release of growth hormone, insulin, and glucagon. (b) Selectivity of intestinal ion transport was achieved by any one of the following alterations: L-alanine substitution at Phenylalanine(11), deletion of Phenylalanine(11), substitution with D-lysine at Lysine(4), or substitution with L-alanine at Lysine(4). These compounds had intestinal ion transport properties of 52, 34, 139, and 94%, respectively, while demonstrating little or no inhibition of growth hormone, insulin or glucagon release. CONCLUSIONS: (a) Phenylalanine(6), Phenylalanine(7), Tryptophan(8), and Lysine(9) are required for the ion transport and other biologic actions of somatostatin, whereas Threonine(10) serves as an essential spacer. (b) Alteration at Phenylalanine(11) or Lysine(4) yields analogs that are selective for ion transport in the rabbit ileum and rat colon. These findings should be taken into consideration when developing a gut-specific somatostatin analog that can be useful in the treatment of diarrhea.
Alanine↗
Mice are insensitive to the antitesticular effects of luteinizing hormone-releasing hormone agonists.
Treatment of male rats with [(imBzl)-D-His6, Pro9-NEt]LHRH or [D-Trp6,Pro9-NEt]LHRH, potent agonists of LHRH, led to a marked decrease in serum testosterone levels and a reduction in testicular LH receptor concentration. Similar treatment of mice showed that they were resistant to the antitesticular effects of the LHRH agonists. To further explore the differences between rats and mice, the direct antitesticular effects of these peptides were investigated in hypophysectomized animals. Hypophysectomized rats and mice were given ovine FSH (50 micrograms), with or without a LHRH agonist (10 micrograms), daily for 5 days. On day 6, the testicular steroidogenic response to hCG was studied. In these studies the in vivo as well as the in vitro steroidogenic response of rat testes to hCG were inhibited by the LHRH analogs. In contrast, pretreatment of mice with the LHRH analogs did not affect their testicular steroidogenic response. Binding studies with the [125I]LHRH analog demonstrated receptors for this peptide on Leydig cells from adult rats. Receptors for LHRH were not, however, detectable on murine Leydig cells. These results suggest that one of the reasons for the lack of an antitesticular effect of LHRH agonists in mice may be due to the inability of these peptides to have a direct effect on testes and may relate to a lack of LHRH receptors.
Animals↗
Effects of synthetic human pancreatic (tumor) GH releasing factor and somatostatin, triiodothyronine and dexamethasone on GH secretion in vitro.
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Animals↗
Effects of synthetic ovine corticotropin-releasing factor, glucocorticoids, catecholamines, neurohypophysial peptides, and other substances on cultured corticotropic cells.
Synthetic ovine corticotropin-releasing factor (CRF) is a 41-residue peptide with high potency and intrinsic activity to stimulate the secretion of ACTH and beta-endorphin-like immunoactivity (beta-End-LI) by cultured adenohypophysial corticotropic cells. The action of CRF in vitro can be potentiated by the weaker secretagogues, vasopressin, oxytocin, epinephrine, norepinephrine, and angiotensin II. CRF-mediated secretion of ACTH and beta-End-LI is noncompetitively inhibited by pretreatment of cells with glucocorticoids. Long term exposure of adenohypophysial cells to CRF results in an increase in total medium plus cell ACTH in the cultures, suggesting that CRF can enhance rates of ACTH synthesis as well as release. CRF also stimulates the secretion of beta-End-LI by corticotropic cells cultured from the neurointermediate lobe. Higher concentrations of CRF are required to stimulate secretion by this cell type than by anterior lobe corticotropic cells. These in vitro results are consistent with CRF playing a major physiological role in the neuroregulation of secretion by anterior lobe corticotropic cells, where the peptide may interact with other modulators.
Adrenocorticotropic Hormone↗
The hormonal actions of corticotropin-releasing factor in sheep: effect of intravenous and intracerebroventricular injection.
The 41-residue ovine corticotropin releasing factor (CRF) was administered iv and intracerebroventricularly (icv) to merino sheep. A significant rise in plasma ACTH, beta-lipotropin (beta LPH) and cortisol was demonstrated after the administration of 200 micrograms, iv. A highly significant correlation between the increments in plasma ACTH and beta LPH was observed. The plasma ACTH rise was evident within 5 min and was abolished by the prior administration of 0.4-4.0 mg dexamethasone. No significant rise in plasma GH, LH, PRL, insulin, glucagon, pancreatic polypeptide, met-enkephalin, angiotensin II, aldosterone, or vasopressin could be demonstrated. Although smaller doses of CRF (50 ng to 5 micrograms) were effective when given icv, the ACTH response was more delayed. It is concluded that CRF stimulates a rapid increase in the secretion of ACTH and beta LPH in sheep. Suppression of this response by dexamethasone indicates that glucocorticoids are capable of acting on the pituitary to inhibit the ACTH response to CRF. The delayed response when CRF is given icv may be due to diffusion. The action of CRF appears to be relatively specific, in that the plasma concentrations of the other pancreatic, pituitary, and adrenal hormones measured were not affected.
Adrenal Cortex Hormones↗
Interaction of corticotropin-releasing factor and arginine vasopressin on adrenocorticotropin secretion in vivo.
A possible interaction between synthetic ovine corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) was tested in anesthetized and in freely moving rats. In animals whose endogenous CRF release was blocked by chlorpromazine-morphine-nembutal, AVP elicited a significantly lower maximum ACTH response than did CRF, whereas the concomitant injection of both peptides resulted in a marked potentiation of CRF-induced ACTH secretion. In freely moving rats, AVP was more potent than CRF (on an equimolar basis) in elevating plasma ACTH levels. Since immunoneutralization of endogenous CRF by the administration of anti-CRF serum significantly reduced ACTH release due to injected AVP in these animals, we suggest that at least part of the AVP-induced ACTH secretion observed in nonanesthetized rats may be due to a potentiation of endogenous CRF by exogenously administered AVP. These data support previous reports of an in vitro synergism between CRF and vasopressin, and emphasize the complex role played by the interaction of these two peptides on ACTH release in vivo.
Adrenocorticotropic Hormone↗