Studies of the role of endothelium-dependent nitric oxide release in the sustained vasodilator effects of corticotrophin releasing factor and sauvagine.
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1. The mechanisms of the sustained vasodilator actions of corticotrophin-releasing factor (CRF) and sauvagine (SVG) were studied using rings of endothelium de-nuded rat thoracic aorta (RTA) and the isolated perfused rat superior mesenteric arterial vasculature (SMA). 2. SVG was approximately 50 fold more potent than CRF on RTA (EC40: 0.9 +/- 0.2 and 44 +/- 9 nM respectively, P < 0.05), and approximately 10 fold more active in the perfused SMA (ED40: 0.05 +/- 0.02 and 0.6 +/- 0.1 nmol respectively, P < 0.05). Single bolus injections of CRF (100 pmol) or SVG (15 pmol) in the perfused SMA caused reductions in perfusion pressure of 23 +/- 1 and 24 +/- 2% that lasted more than 20 min. 3. Removal of the endothelium in the perfused SMA with deoxycholic acid attenuated the vasodilatation and revealed two phases to the response; a short lasting direct action, and a sustained phase which was fully inhibited. 4. Inhibition of nitric oxide synthase with L-NAME (100 microM) L-NMMA (100 microM) or 2-ethyl-2-thiopseudourea (ETPU, 100 microM) had similar effects on the vasodilator responses to CRF as removal of the endothelium, suggesting a pivotal role for nitric oxide. However the selective guanylate cyclase inhibitor 1H-[l,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 10 microM) did not affect the response to CRF. 5. High potassium (60 mM) completely inhibited the vasodilator response to CRF in the perfused SMA, indicating a role for K channels in this response. 6. Compared to other vasodilator agents acting via the release of NO, the actions of CRF and SVG are strikingly long-lasting, suggesting a novel mechanism of prolonged activation of nitric oxide synthase. (+info)
Urocortin is the principal ligand for the corticotrophin-releasing factor binding protein in the ovine brain with no evidence for a sauvagine-like peptide.
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To purify novel ligands for the corticotrophin-releasing factor binding protein (CRF-BP) from ovine brain, whole brain was homogenised in methanol and the supernatant extracted on Sep-pak C18 cartridges followed by a preliminary HPLC step. Three peaks of ovine CRF-BP ligand activity were detected in the HPLC fractions, the first two of which were also detected by a specific corticotrophin-releasing factor two-site immunoradiometric assay, the third peak being detected by a human CRF-BP ligand assay, which will not detect ovine CRF. Human CRF-BP ligand-containing fractions were further purified by affinity chromatography on a human recombinant CRF-BP column with two additional HPLC steps. The human CRF-BP ligand was found to: (a) possess a molecular mass of 4707 Daltons, (b) have an N-terminal amino acid sequence (5 residues) identical to rat urocortin, (c) be detected by a specific urocortin radioimmunoassay, (d) have high affinity for both the human and ovine CRF-BPs and (e) be present in many regions of the ovine brain. Additionally, a 300 bp cDNA fragment sharing 83% homology with the rat urocortin gene was cloned from ovine brain, the product of which was predicted to have an identical amino acid sequence to that of rat urocortin. These pieces of information confirmed the identity of the human CRF-BP ligand as an ovine urocortin. The specially developed CRF-BP ligand assays showed that the rank orders of affinity of the CRF family members for human CRF-BP were: carp urotensin-1>>human CRF=rat/ovine urocortin>human urocortin>>frog sauvagine>>ovine CRF, and those for the ovine CRF-BP were: carp urotensin-1> human CRF=rat/ovine urocortin>human urocortin> frog sauvagine>>ovine CRF. This study describes a successful technique for the purification and detection of peptide ligands for the CRF-BP. We conclude that urocortin is the principal ligand for the CRF-BP in ovine brain and we could find no evidence for a centrally located mammalian sauvagine-like peptide. (+info)
Purification and characterization of rat des-Gln14-Ghrelin, a second endogenous ligand for the growth hormone secretagogue receptor.
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Ghrelin, a peptide purified from the stomach, is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and potently stimulates growth hormone release from the pituitary. Ghrelin is modified with an n-octanoyl group at Ser(3). This modification is essential for the activity of ghrelin. Previously, it was not known whether other ligands for GHS-R existed. Here, we report the purification of the second endogenous ligand for GHS-R from rat stomach. This ligand, named des-Gln(14)-ghrelin, is a 27-amino acid peptide, whose sequence is identical to ghrelin except for one glutamine. Southern blotting analysis under low hybridization conditions indicates that no homologue for ghrelin exists in rat genomic DNA. Furthermore, genomic sequencing and cDNA analysis indicate that des-Gln(14)-ghrelin is not encoded by a gene distinct from ghrelin but is encoded by an mRNA created by alternative splicing of the ghrelin gene. This is the first example of a novel mechanism that produces peptide multiplicity. Des-Gln(14)-ghrelin has an n-octanoyl modification at Ser(3) like ghrelin, which is also essential for its activity. Des-Gln(14)-ghrelin-stimulated growth hormone releases when injected into rats. Thus, growth hormone release is regulated by two gastric peptides, ghrelin and des-Gln(14)-ghrelin. (+info)
Ghrelin elicits a marked stimulatory effect on GH secretion in freely-moving rats.
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Ghrelin is a growth hormone-releasing acylated peptide from stomach. The purified peptide consist of 28 amino acids in which the serine 3 residue is n-octanoylated. Ghrelin has been reported to increase in vitro GH secretion as well as in vivo plasma GH levels in pentobarbital anaesthetized rats. The aim of this work was to characterize the stimulatory effect of Ghrelin on in vivo GH secretion in freely-moving rats. Furthermore, we compare the effect of Ghrelin with GHRH. In addition to vehicle, we administered different doses of Ghrelin (3 nmol/Kg, 12 nmol/Kg and 60 nmol/Kg); GHRH (3 nmol/Kg and 12 nmol/kg). Plasma GH levels were measured in blood samples taken at 5, 10, 15, 20, 30 and 45 min after their administration as an i.v. bolus at 0 min. Administration of Ghrelin led to an increase in plasma GH levels at all time-points tested (5, 10, 15, 20 and 30 min, P<0.01; and 45 min, P<0.05) in comparison to control untreated rats. A maximal stimulatory effect on plasma GH was observed following administration of 12 nmol/Kg of Ghrelin, the effect being similar to the one obtained with 60 nmol/Kg in terms of both AUC and mean peak GH levels. At the dose of 3 nmol/Kg GHRH and Ghrelin exhibited a similar stimulatory effect in term of both, AUC and mean peak GH levels. However following administration of a dose of 12 nmol/Kg, the effect of Ghrelin was much greater than the same dose of GHRH in terms of both AUC and mean peak GH levels. In summary, this study provides the first evidences that Ghrelin exert a marked stimulatory effect in plasma GH levels in freely-moving rats and provides further evidences that Ghrelin may play an important role in the physiological control of GH secretion. (+info)
Kidney produces a novel acylated peptide, ghrelin.
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Ghrelin is a novel growth hormone-releasing peptide with a unique acylated structure. Here we reveal that prepro-ghrelin gene is expressed in the mouse kidney and glomerulus. We also show by reverse-phase high performance liquid chromatography coupled with radioimmunoassay that the mouse kidney does produce ghrelin. The ghrelin immunoreactivity in the mouse kidney is 6.79+/-0.48 fmol/mg (n=5), which is much more abundant than that in the mouse plasma of 0.339+/-0.029 fmol/microl (n=6). Furthermore, prepro-ghrelin gene is expressed in cultured rat mesangial cells, fibroblast-like NRK-49F cells and mouse podocytes, but not in rat epithelial cell-like NRK-52E cells. Ghrelin receptor gene is also expressed in the rat kidney. These findings demonstrate that the kidney, glomerulus and renal cells express prepro-ghrelin gene and ghrelin is produced locally in the kidney, and suggest the endocrine and/or paracrine roles of ghrelin in the kidney. (+info)
Ghrelin-induced growth hormone secretion in humans.
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Ghrelin is a novel growth hormone (GH) releaser acylated peptide that has recently been purified from stomach, and which potently binds to the GH secretagogue receptor. Ghrelin releases GH in vitro and in vivo in animal models, however its actions, potency and specificity in humans are unknown. In the present study, 12 healthy subjects were studied: 6 underwent four tests with ghrelin administered i.v. at the dose of 0 (placebo), 0.25, 0.5 and 1 microg/kg which corresponds to 0, 18, 37 and 75 microg total dose. A further 6 volunteers underwent two tests on different days with ghrelin at the dose of 3.3 or 6.6 microg/kg which corresponds to 250 microg and 500 microg total dose. Ghrelin-mediated GH secretion showed a dose-response curve, in which 1 microg/kg was the minimally effective dose in some individuals, but not as a group. On the contrary, the total doses of 250 microg and 500 microg elicited a powerful GH secretion, with a mean peak of 69.8+/-9.2 microg/l and 90.9+/-16.9 microg/l respectively, and areas under the curve of 4435+/-608 and 6125+/-1008 microg/l per 120 min respectively. All of them statistically significant vs placebo and vs the 1 microg/kg dose. Ghrelin administration also elicited a relevant dose-response mediated prolactin secretion suggesting no specificity of its actions. No relevant side effects were observed with ghrelin apart from a hyperhydrosis episode in two individuals tested with the higher ghrelin doses. In conclusion, ghrelin is a potent releaser of GH in normal individuals, with a dose-response pattern of operation. No saturating dose was observed. (+info)
Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway.
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Ghrelin, an endogenous ligand for growth hormone secretagogue (GHS) receptor originally isolated from the stomach, occurs in the hypothalamic arcuate nucleus and may play a role in energy homeostasis. Synthetic GHSs have activated the hypothalamic arcuate neurons containing neuropeptide Y (NPY), suggesting the involvement of NPY in some of ghrelin actions. This study was designed to elucidate the role of ghrelin in the regulation of food intake. A single intracerebroventricular (ICV) injection of ghrelin (5-5,000 ng/rat) caused a significant and dose-related increase in cumulative food intake in rats. Ghrelin (500 ng/rat) was also effective in growth hormone-deficient spontaneous dwarf rats. Hypothalamic NPY mRNA expression was increased in rats that received a single ICV injection of ghrelin (500 ng/rat) (approximately 160% of that in vehicle-treated groups, P < 0.05). The ghrelin's orexigenic effect was abolished dose-dependently by ICV co-injection of NPY Y1 receptor antagonist (10-30 microg/rat). The leptin-induced inhibition of food intake was reversed by ICV co-injection of ghrelin in a dose-dependent manner (5-500 ng/rat). Leptin reduced hypothalamic NPY mRNA expression by 35% (P < 0.05), which was abolished by ICV co-injection of ghrelin (500 ng/rat). This study provides evidence that ghrelin is an orexigenic peptide that antagonizes leptin action through the activation of hypothalamic NPY/Y1 receptor pathway. (+info)
Circulating ghrelin levels are decreased in human obesity.
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Ghrelin is a novel endogenous natural ligand for the growth hormone (GH) secretagogue receptor that has recently been isolated from the rat stomach. Ghrelin administration stimulates GH secretion but also causes weight gain by increasing food intake and reducing fat utilization in rodents. To investigate the possible involvement of ghrelin in the pathogenesis of human obesity, we measured body composition (by dual X-ray absorption) as well as fasting plasma ghrelin concentrations (radioimmunoassay) in 15 Caucasians (8 men and 7 women, 31+/-9 years of age, 92+/-24 kg body wt, and 29+/-10% body fat, mean +/- SD) and 15 Pima Indians (8 men and 7 women, 33+/-5 years of age, 97+/-29 kg body wt, and 30+/-8% body fat). Fasting plasma ghrelin was negatively correlated with percent body fat (r = -0.45; P = 0.01), fasting insulin (r = -0.45; P = 0.01) and leptin (r = -0.38; P = 0.03) concentrations. Plasma ghrelin concentration was decreased in obese Caucasians as compared with lean Caucasians (P < 0.01). Also, fasting plasma ghrelin was lower in Pima Indians, a population with a very high prevalence of obesity, compared with Caucasians (87+/-28 vs. 129+/-34 fmol/ml; P < 0.01). This result did not change after adjustment for fasting plasma insulin concentration. There was no correlation between fasting plasma ghrelin and height. Prospective clinical studies are now needed to establish the role of ghrelin in the pathogenesis of human obesity. (+info)