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Background and purpose: Ghrelin, a gut-brain peptide, is considered a gastroprotective factor in gastric mucosa. We investigated the role of prostaglandins (PG) and the possible interplay between PGs and nitric oxide (NO) in ghrelin gastroprotection against ethanol (EtOH)-induced gastric lesions. EXPERIMENTAL APPROACH: We examined the effects of (1) central ghrelin (4 mug per rat) injection on PGE(2) accumulation in normal or EtOH-lesioned gastric mucosa, (2) pretreatment with indomethacin (10 mg kg(-1), p.o.), a non-selective cyclooxygenase (COX) inhibitor, and with a selective COX-1, SC560 (5 mg kg(-1), p.o.) or COX-2 inhibitor, celecoxib (3.5 mg kg(-1), p.o.) on ghrelin gastroprotection against 50% EtOH (1 mL per rat)-induced gastric lesions, (3) the NO synthase inhibitor, L-NAME (70 mg kg(-1), s.c), on gastric PGE(2) content in ghrelin-treated rats and (4) central ghrelin on the expression of constitutive and inducible NOS and COX mRNA and on the localization of the immunoreactivity for COX-2 in the gastric mucosa exposed to EtOH. KEY RESULTS: Ghrelin increased PGE(2) in normal mucosa, whereas, it reversed the EtOH-induced PGE(2) surge. Ghrelin had no effect on mucosal COX-1 expression but reduced the EtOH-induced increase in COX-2 expression and immunoreactivity. Indomethacin and SC560, but not celecoxib, removed ghrelin gastroprotection. L-NAME prevented the PGE(2) surge induced by ghrelin and, like indomethacin, reduced EtOH-induced PGE(2) increase. Ghrelin enhanced eNOS expression and reduced iNOS mRNA. CONCLUSIONS AND IMPLICATIONS: This study shows that COX-1-derived PGs are mainly involved in ghrelin gastroprotection and that the constitutive-derived NO together with PGE(2) are involved in ghrelin gastroprotective activity.

It has been extensively demonstrated that GH secretagogues (GHS) play a role in the regulation of bone metabolism in animals and humans. Unlike GH, administration of GHS does not increase bone resorption markers, suggesting that a mechanism exclusively linked to GH release cannot account for the effect of these compounds. On this line, we investigated the effect of GHS and ghrelin, the endogenous ligand of GHS receptors, on bone cells. We found that both hexarelin and ghrelin significantly stimulated cell proliferation and increased alkaline phosphatase and osteocalcin production in primary cultures of rat calvaria osteoblasts. In the same cells, we were able to detect the mRNA for the GHS receptor by RT-PCR and the corresponding protein by Western blot, indicating that ghrelin and GHS may bind and activate this receptor. Two isoforms of GHS receptors (GHS-R), which are presumably the result of alternate processing of pre-mRNA, have been identified and designed receptors 1a (R1a) and 1b (R1b). Ghrelin, the endogenous ligand of the GHS receptors, binds with high affinity GHS-R1a only. Unlike fetal calvaria cells, osteoblasts derived from adult rat tibia did not express the GHS-R1 a, but only the biologically inactive isoform GHS-R1b. The latter isoform was present in only one of the three specimens of human osteoblasts obtained from the iliac crest or the upper femur of patients during surgery. These results would indicate that only osteoblasts from fetal bone express functional receptors responsive to ghrelin and GHS.

Ghrelin, an acylated peptide produced predominantly by the stomach, has been discovered to be a natural ligand of the growth hormone secretagogue receptor 1a (GHS-R1a). It is localized in distinct cells of the gastric mucosa, mainly distributed in the mid portion of the oxyntic gland characterized by P/D1 granules in man and X/A-like granules in rodents. The ghrelin cell represents the second most frequent endocrine cell type after the enterochromaffin-like cells in gastric oxyntic mucosa, pointing to a potentially relevant role in the physiology of the stomach. Ghrelin has no relevant homology with any known gastrointestinal peptide and displays strong GH-releasing activity both in animals and in humans. However, in addition to stimulating GH secretion, ghrelin possesses several other endocrine and extraendocrine biological activities that are explained by the widespread distribution of ghrelin and GHSR1a expression. In the rat, ghrelin exerts a control in gastric acid secretion and motility: the gastric acid secretion is stimulated by peripheral administration of high doses of ghrelin, but inhibited by very low doses of ghrelin delivered into the central nervous system. Moreover, ghrelin provides a potent and dose-related gastroprotective action against ethanol- and stress-induced gastric ulcers. The integrity of both nitric oxide (NO) system and capsaicin afferent nerves are required for the gastroprotective effect of ghrelin, whereas the vagus nerve might be involved in conveying ghrelinergic signal from periphery to the brain. In addition, prostaglandins derived by the constitutive cyclooxygenase (COX) activity are essential for the protective activity of ghrelin in ethanol and stress-induced gastric lesions. Given its prevailing role in physiological and pathophysiological gastric function, the discovery of ghrelin will open new perspectives and potential clinical implications in the gastroenteric field.

The possibility of changes in binding sites for calcitonin gene-related peptide (CGRP) in response to sensorial denervation was studied in rats after pyridoxine-induced neurotoxicity. Changes in CGRP binding sites were evaluated by an in vitro autoradiographic technique using [125I]-Tyr-rat-CGRP as a ligand. A bidirectional binding response was obtained: an increased binding, in comparison with the respective control, was observed in spinal cord and cerebellar regions whereas a decreased binding was noted in cerebrum. CGRP binding in selective areas comparising the pathways that are responsible for transmitting sensory impulses have shown significant changes. These results suggest that CGRP plays a modulatory role in the central nervous system. This study has also proposed and evaluated pyridoxine toxicity as a model for studying sensory denervation.

We demonstrate both simultaneous supercontinuum generation and efficient phase-matched UV generation around 350nm in a highly nonlinear silica holey fiber. The resulting spectrum extends from 1800nm and, as far as we are aware, is the broadest optical spectrum yet reported for a fiber based supercontinuum generator.

Ghrelin, the endogenous ligand of the GH secretagogue receptor (GHS-R) has been previously shown to inhibit gastric acid secretion in pylorus-ligated rats. Two isoforms of GHS-R have been identified: GHS-R(1a) and GHS-R(1b). The present study aimed: (i) to characterise the type of GHS-R involved in the central gastric inhibitory activity of ghrelin by using des-octanoyl ghrelin, and synthetic GHS-R(1a) agonist (EP1572) and antagonist (D-Lys(3)-GHRP-6) and (ii) to investigate the relationship between ghrelin and cortistatin (CST) in the control of gastric acid secretion by using the natural neuropeptide CST-14 and the synthetic octapeptide CST-8. The specific interactions of all the compounds with GHS-R(1a) were determined by comparing their ability to displace labelled ghrelin or somatostatin from its receptors on rat hypothalamic membranes or on rat cardiomyocyte, respectively. Intracerebroventricular administration of 0.01 and 1 nmol/rat des-octanoyl ghrelin did not affect gastric acid secretion in pylorus-ligated rats, whereas EP1572 either i.c.v. (0.01-1 nmol/rat) or i.p. (10 and 20 nmol/kg) inhibited acid gastric secretion. Preteatment with D-Lys(3)GHRP-6 (3 nmol/rat, i.c.v.) was able to remove the inhibitory action of ghrelin (0.01 nmol/rat, i.c.v.) on gastric acid volume and acid output, thus indicating that the type 1a GHS-R likely mediates the gastric inhibitory action of ghrelin. This is supported by binding data showing that D-Lys(3)GHRP-6, but not des-octanoyl ghrelin, binds to hypothalamic GHS-R. CST-14 (1 nmol/rat, i.c.v.) did not affect either basal or ghrelin inhibition of gastric acid secretion. CST-8 (1 nmol/rat, i.c.v.) was able to counteract the gastric ghrelin response. The observation that CST-14 binds both GHR-S and somatostatin receptors, whereas CST-8 specifically displaces only ghrelin binding, indicates that CST-8 behaves as a GHS-R(1a) antagonist.

We examined the possible central and peripheral effects of synthetic growth hormone secretagogues (GHS), hexarelin (Hexa) and EP 40737 (D-Thr-D-Trp (2-Me)-Ala- Trp-D-Phe-Lys-NH2), and of their endogenous counterpart, ghrelin, on gastric acid secretion. The compounds were administered intracerebroventricularly (i.c.v.) or subcutaneously (s.c.) in conscious male rats and the volume of gastric secretion and gastric acid output were examined 3 h after pylorus ligation (Shay-test). Central Hexa, EP 40737 and ghrelin administration (from 0.1 pmol to 1 nmol/rat, i.c.v.) significantly inhibited gastric acid secretion. The maximum inhibitory effect on gastric acid output was detected at the dose of 10 pmol/rat, i.c.v. for Hexa (–51.3%), of 100 pmol/rat, i.c.v. for EP 40737 (–70%) and of 1 pmol/rat, i.c.v. for ghrelin (–60%). All peptides were less effective at the highest dose used (1 nmol/rat, i.c.v.). Hexa, EP 40737 and ghrelin injected s.c. did not modify gastric acid secretion. The inhibitory action of Hexa on gastric acid secretion seems to involve brain somatostatinergic system since Hexa (10 pmol/rat, i.c.v.) did not inhibit gastric acid secretion in rats pretreated (4 h before) with cysteamine (300 mg/kg, s.c.), a depletor of endogenous somatostatin. These results show that synthetic GHS and ghrelin exert a central long-lasting inhibitory effect on gastric acid secretion in conscious pylorus-ligated rats. The fact that very low doses of ghrelin and GHS inhibit gastric secretion, provide evidence for a tonic inhibitory role of the peptides in the central control of gastric secretory function.

This study examined the effects of intracerebroventricular (i.c.v.), intraperitoneal (i.p.) or intraplantar (i.pl.) administration of ghrelin, the endogenous ligand of the growth hormone secretagogue receptor, in the development of hyperalgesia and edema induced by intraplantar injection of carrageenan in rats. Central ghrelin (4 ng to 4 microg/rat) given 5 min before carrageenan produced a dose-related reversal of carrageenan-induced mechanical hyperalgesia measured by Randall-Selitto test with an ED50 of 81.7 ng/rat. Ghrelin at the dose of 4 microg/rat i.c.v. was also effective in inhibiting edema. When ghrelin (4 microg/rat i.c.v.) was administered 150 min after carrageenan, it failed to modify either hyperalgesia or the paw volume. Given i.p., 30 min before carrageenan, ghrelin (20-160 microg/kg) induced a significant dose-dependent inhibition of hyperalgesia with an ED50 of 77 microg/kg and a slight reduction of edema. Intraplantar ghrelin (40 ng to 12 microg/rat) did not significantly modify both the hyperalgesic and edematous activities of carrageenan. The anti-hyperalgesic and anti-edematous effects of ghrelin (4 microg/rat i.c.v.) were reversed by naloxone (10 microg/rat i.c.v.). Systemic administration of the peripheral selective opioid antagonist, naloxone methiodide (3 mg/kg s.c.), did not antagonize antinociception elicited by i.p. ghrelin. Overall these data indicate that ghrelin exerts an inhibitory role on inflammatory pain through an interaction with the central opioid system.

We studied the effect of the acute central administration of obestatin on food intake and body weight in short-term starved male rats, and those of 28-day continuous intracerebroventricular (icv) infusion of obestatin in free feeding rats. In 16-h starved rats, obestatin induced a trend toward a reduction of food intake that did not reach statistical significance. In fed rats, the icv infusion of obestatin significantly decreased food consumption in the first day of treatment; but the anorexigenic effect of obestatin vanished thereafter. Interestingly, the body weight of rats infused for 28 days with obestatin was superimposable to that of the respective control at all time intervals. In all, our results indicate that the anorexigenic effect of obestatin is of little account and that the peptide does not modify energy metabolism in the long-term administration.

It has previously been reported that growth hormone secretagogues (GHS) may have a role in the regulation of bone metabolism in animals and humans. In this study we evaluated the effect of ghrelin, the endogenous ligand of GHS receptors, on the proliferation rate and on osteoblast activity in primary cultures of rat calvaria osteoblasts. In the same experiments, we compared the effects of ghrelin with those of hexarelin (HEXA) and EP-40737, two synthetic GHS with different characteristics. Both ghrelin and HEXA (10−11−10−8 M) significantly stimulated osteoblast proliferation at low concentrations (10−10 M). Surprisingly, EP-40737 demonstrated an antiproliferative effect at 10−9−10−8 M, whereas lower concentrations had no effect on cell proliferation. Ghrelin and HEXA significantly increased alkaline phosphatase (ALP) and osteocalcin (OC) production. At variance with these peptides, EP-40737 did not significantly stimulate ALP and OC. The mRNA for GHS-R1a receptors and the corresponding protein were detected in calvarial osteoblasts by RT-PCR and Western blot respectively, indicating that ghrelin and GHS may bind and activate this specific receptor. We conclude that endogenous ghrelin and synthetic GHS modulate proliferation and differentiation of rat osteoblasts, probably by acting on their specific receptor.

The effects of intracerebroventricular (icv) or subcutaneous (sc) hexarelin (Hexa) administration. against gastric ulcers induced by ethanol (50%, 1 ml/rat/os) or Indomethacin (20 mg/kg/os) were examined in conscious rats. Hexa at 1 nmol/rat, icv or 10 nmol/kg, sc reduced ethanol-induced ulcers by 47% and 32% respectively. Hexa, but not ghrelin significantly worsened (+40%) Indomethacin-induced ulcers when injected sc. Hexa-gastroprotection against ethanol-induced ulcers was removed by the GHS-R antagonist (D-Lys(3))-GRPR-6 and by the inhibitor of NO-synthase (NOS) N-omega-nitro-L-arginine methyl ester. Semiquantitative RT-PCR assay of gastric NOS mRNA isoforms revealed that the reduction in iNOS-derived NO and the increase of constitutive-derived NO are relevant for the gastroprotection of Hexa against ethanol-induced gastric damage. (C) 2004 Elsevier Inc. All rights reserved.

Ghrelin, the endogenous ligand for GH secretagogue receptors, has been reported to influence acid gastric secretion and motility, but its potential gastroprotective effect is unknown. The aims of this study were 1) to examine the effects of central and peripheral administration of ghrelin on ethanol-induced gastric ulcers in conscious rats, and 2) to investigate the possible roles of nitric oxide (NO), vagal nerve, and sensory fibers in the gastric effects of ghrelin. Ghrelin was administered either intracerebroventricularly or sc 30 min before ethanol, and mucosal lesions were examined macroscopically. Additionally, rats were either treated with the inhibitor of NO synthesis Nω-nitro-l-arginine methyl ester (L-NAME) or underwent bilateral cervical vagotomy or capsaicin-induced sensory denervation. Conventional histology and immunohistochemistry for ghrelin, gastrin, and somatostatin were performed on gastric specimens from representative rats. Central ghrelin (4–4000 ng/rat) dose-dependently reduced ethanol-induced gastric ulcers by 39–77%. Subcutaneous ghrelin administration (80 μg/kg) reduced ulcer depth only. L-NAME and capsaicin, but not vagotomy, prevented the gastroprotective effect of central ghrelin (4000 ng/rat). This is the first evidence that ghrelin exerts a potent central gastroprotective activity against ethanol-induced lesions. The gastroprotective effect of ghrelin is mediated by endogenous NO release and requires the integrity of sensory nerve fibers.

To study possible age-related differences in the role of neuronal histaminergic pathways in the control of GH secretion, the effects of α-fluoromethylhistidine (α-FMH), an irreversible inhibitor of histamine (HA) synthesis, were examined on basal and opioid-induced GH release in neonatal and adult rats. The mechanisms involved in such effects were evaluated by measuring pituitary GH mRNA levels and hypothalamic levels of GH-releasing hormone (GHRH) and somatostatin (SRIF) mRNAs. Daily injection of α-FMH (20 mg/kg, s.c.) in pups of either sex, from birth until 10 days of age, caused a significant increase in baseline plasma GH and potentiated the GH response to the [Met5]-enkephalin analog FK 33–824 (1 mg/kg, s.c.) administered 3 h after the last α-FMH injection. GH and SRIF mRNA levels were significantly higher in α-FMH-treated pups than in controls, whereas no difference was observed in GHRH mRNA levels. In young adult male rats, acute administration of α-FMH (100 mg/kg, s.c., 3 h before) did not change significantly basal GH levels but potentiated FK 33–824 (0·3 mg/kg, intracarotid)-induced stimulation of GH secretion. Repeated administration of α-FMH (200 μg/rat, i.c.v., for 3 days) failed to modify basal and FK 33–824-induced GH secretion, caused a significant reduction in hypothalamic GHRH mRNA levels and left SRIF and GH mRNAs unchanged. These findings indicate that HA exerts an inhibitory effect on GH secretion in both neonatal and adult rats. The different effects of short-term HA depletion on hypothalamic and pituitary indices of somatotropic function observed at the two age periods may be ascribed to the immaturity of the HA system in early postnatal life and to a different functional role of GH-regulatory factors during ontogeny. Journal of Endocrinology (1996) 151, 195–201