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Kenji Kangawa

Publications and source records attributed to Kenji Kangawa.

At least 199 records · Page 11Linked to original sources
PubMed2003

Ghrelin-induced food intake is mediated via the orexin pathway.

The hypothalamus regulates energy intake by integrating the degree of starvation or satiation with the status of the environment through a variety of neuronal and blood-derived signals. Ghrelin, a peptide produced in the stomach and hypothalamus, stimulates feeding and GH secretion. Centrally administered ghrelin exerts an orexigenic activity through the neuropeptide Y (NPY) and agouti-related protein systems. The interaction between ghrelin and other hypothalamic orexigenic peptides, however, has not been clarified. Here, we investigated the anatomical interactions and functional relationship between ghrelin and two orexigenic peptides, orexin and melanin-concentrating hormone (MCH), present in the lateral hypothalamus. Ghrelin-immunoreactive axonal terminals made direct synaptic contacts with orexin-producing neurons. Intracerebroventricular administration of ghrelin induced Fos expression, a marker of neuronal activation, in orexin-producing neurons but not in MCH-producing neurons. Ghrelin remained competent to induce Fos expression in orexin-producing neurons following pretreatment with anti-NPY IgG. Pretreatment with anti-orexin-A IgG and anti-orexin-B IgG, but not anti-MCH IgG, attenuated ghrelin-induced feeding. Administration of NPY receptor antagonist further attenuated ghrelin-induced feeding in rats treated with anti-orexin-IgGs. Ghrelin-induced feeding was also suppressed in orexin knockout mice. This study identifies a novel hypothalamic pathway that links ghrelin and orexin in the regulation of feeding behavior and energy homeostasis.

Animals↗
PubMed2003

Gene expression, secretion, and autocrine action of C-type natriuretic peptide in cultured adult rat cardiac fibroblasts.

C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is known to be synthesized in the central nervous system and vascular endothelial cells, in contrast to atrial natriuretic peptide and brain natriuretic peptide. However, there have been no studies concerning CNP production in cultured cardiac cells. Here, we examined the production and the local effect of CNP in cultured ventricular cells. Under serum-free conditions, adult rat cardiac fibroblasts secreted immunoreactive CNP time dependently. TGF-beta1, basic fibroblast growth factor, and endothelin-1 significantly stimulated CNP secretion. Northern blot analysis detected significant expressions of CNP and its specific receptor (guanylyl cyclase-B) mRNA in cardiac fibroblasts. CNP stimulated intracellular cGMP production in fibroblasts more intensely than atrial and brain natriuretic peptides. CNP inhibited both DNA and collagen syntheses of cardiac fibroblasts, and these inhibitory effects by CNP were stronger than by atrial and brain natriuretic peptides. The inhibition by CNP of DNA and collagen syntheses was reproduced by a cGMP analog, 8-bromo cGMP. The present findings demonstrate that CNP is synthesized in and secreted from cardiac fibroblasts and suggest that CNP has a suppressive effect on fibroblast proliferation and extracellular matrix production, probably via the guanylyl cyclase-B-mediated cGMP-dependent process. CNP produced by cardiac fibroblasts may play a role as an autocrine regulator against excessive cardiac fibrosis.

Age Factors↗
PubMed2003

Ghrelin is involved in the decidualization of human endometrial stromal cells.

Successful implantation involves a complex interaction between the endometrium and the embryo. It is well known that several neuropeptides are expressed in the endometrium and placenta during embryonal implantation, suggesting an important role as chemical mediators of the feto-maternal relationship. Ghrelin has recently been identified as the endogenous ligand for the GH secretagogue receptor. Ghrelin is a peptide hormone with many physiological functions, and its expression in the human placenta has been reported. To investigate the involvement of ghrelin in embryonal implantation, we assessed the spatio-temporal expression pattern of ghrelin and its receptor in the human endometrium and placenta through the normal menstrual cycle and in early pregnancy. We also examined the effect of ghrelin on the decidualization of endometrial stromal cells (ESC). Weak expression of ghrelin mRNA was detected in the nonpregnant endometrium, and it was dramatically increased in the decidualized endometrium. A GH secretagogue receptor mRNA was detected in the endometrium throughout the normal menstrual cycle and in early pregnancy, but not in the first trimester placenta. Immunohistochemical analysis using an antighrelin antibody revealed strong signals in decidual cells and extravillous trophoblast cells. Coculture with first trimester placenta up-regulated ghrelin mRNA expression by primary cultured ESC, although sex steroids and 8-bromo-cAMP had no effect. In addition, ghrelin enhanced the decidualization of ESC induced by 8-bromo-cAMP (8-Br-cAMP) in vitro. Thus, ghrelin is a novel paracrine/autocrine factor that is involved in cross-talk between the endometrium and embryo during embryonal implantation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2003

Ghrelin concentration in cord and neonatal blood: relation to fetal growth and energy balance.

To investigate the relationship between ghrelin and both fetal and neonatal growth parameters and energy balance, we measured plasma ghrelin concentrations in 54 cord blood samples (male, n = 34; female, n = 20; gestational age, 37.0-41.6 wk; birth weight, 2206-4326 g) and 47 neonatal blood samples (male, n = 27; female, n = 20; postnatal d 3-8). The plasma ghrelin concentrations in cord blood ranged from 110.6-446.1 pmol/liter (median, 206.7 pmol/liter), which were equal to or higher than those in normal weight adults. These values were inversely correlated with birth weight (r = -0.40; P = 0.002), birth length (r = -0.36; P = 0.007), placental weight (r = -0.35; P = 0.01), and IGF-I concentration (r = -0.49; P = 0.0002), but were not significantly correlated with the GH concentration (r = 0.22; P = 0.12). The ghrelin concentrations in small for gestational age newborn were significantly higher than those in appropriate for gestational age newborns (P = 0.0008). The ghrelin concentrations in the vein were significantly higher than those in the artery in 8 cord blood samples (P = 0.01), which suggests that the placenta is an important source of fetal ghrelin. In neonates, the ghrelin concentrations ranged from 133.0-481.7 pmol/liter (median, 268.3 pmol/liter), which were significantly higher than those in cord blood (P < 0.0001). These results suggest that ghrelin may contribute to fetal and neonatal growth.

Birth Weight↗
PubMed2003

Cardioprotective effect of adrenomedullin in heart failure.

Many neurohumoral factors participate in the pathophysiology of heart failure, and adrenomedullin (AM) may be involved in their derangement. This work reviews the accumulating evidence in support of a compensatory role of AM in heart failure, and describes the possible mechanisms of this role. It has been established that plasma AM levels are increased in patients with heart failure in proportion to the severity of the disease. Furthermore, recent studies suggest that plasma AM level is an independent prognostic indicator of heart failure. Thus, AM may be not only a biochemical marker for evaluating the severity of heart failure, but also a prognostic indicator of this syndrome. In patients with heart failure, AM production is increased not only in the plasma, but also in the heart. AM secretion from the failing human heart is also increased, but this increase is small and responds slowly to the stimulus. This phenomenon may be explained by the fact that AM is secreted via a constitutive pathway and that AM is an autocrine and/or a paracrine factor in the heart. An experiment using cultured myocytes suggested that cytokines and mechanical stress are important stimuli for AM production in the heart. Regarding the action of AM in the heart, recent studies have suggested that AM exerts an inotropic action both in vitro and in vivo. AM also attenuates cardiac hypertrophy in myocytes and inhibits proliferation and collagen production in cardiac fibroblasts. These results suggest that AM may be an antifibrotic, antihypertrophic, and positive inotropic factor in the failing and hypertrophied heart. Because AM has many cardiorenal actions, AM administration may be useful for the treatment of heart failure. Indeed, acute administration of AM has been shown to improve the hemodynamics, renal function, and hormonal parameters in patients with heart failure. Moreover, recent studies have shown that AM gene therapy or long-term AM infusion significantly improved cardiac hypertrophy and fibrosis, and prolonged the survival time in an animal model of hypertension and heart failure. In conclusion, these findings suggest that AM plays a compensatory role in the pathophysiology of heart failure and that administration of AM may be a new and promising approach for the treatment of patients with this syndrome.

Adrenomedullin↗
PubMed2003

Pulmonary vasodilator response to adrenomedullin in patients with pulmonary hypertension.

This study sought to investigate pulmonary vasodilator responses to intrapulmonary and intravenous infusion of adrenomedullin (AM) in patients with pulmonary hypertension. In 10 patients with pulmonary hypertension, blood flow velocity in a segmental pulmonary artery was measured using a Doppler flow wire during intrapulmonary infusion of AM, acetylcholine (ACh), and adenosine triphosphate (ATP). The hemodynamic effects of intravenously administered AM (0.05 microg/kg/min) were examined in another 5 patients with primary pulmonary hypertension. Intrapulmonary infusion of AM, ACh or ATP caused a significant dose-dependent increase in blood flow velocity in a segmental pulmonary artery, respectively. The increase in flow velocity with AM at 10(-8) mol/l (41 +/- 6% of the baseline value) was comparable to that with ACh at 10(-4) mol/l (39 +/- 11%) and that with ATP at 10(-5) mol/l (36 +/- 14%), suggesting a strong pulmonary vasodilator activity of AM. Intravenous infusion of AM produced a 41% increase in cardiac index (p < 0.05) and a 30% decrease in pulmonary vascular resistance (p < 0.05) with a 3% reduction in mean pulmonary arterial pressure (p = NS). These results suggest that, on a molar basis, AM may have much more potent pulmonary vasodilator activity than ACh and ATP, and thus may have beneficial hemodynamic effects in patients with pulmonary hypertension.

Acetylcholine↗
PubMed2003

Direct measurement of glycine-extended adrenomedullin in plasma and tissue using an ultrasensitive immune complex transfer enzyme immunoassay in rats.

The mature form of the vasodilator peptide adrenomedullin (AM-m) is synthesized from a glycine-extended precursor (AM-Gly) by enzymatic amidation. We have developed a highly sensitive enzyme immunoassay (Immune Complex Transfer Enzyme Immunoassay; ICTEIA) that enables us to measure levels of AM-Gly in plasma and tissue directly. The detection limit of this assay is 1 amol/assay, and the intra- and inter-assay precision are 4.5-14.1% and 9.9-20.5%, respectively. Dilution curves for plasma samples showed good linearity, and the analytical recovery was 107-116.6%. Using ICTEIA, we determined that the plasma concentration of immunoreactive AM-Gly is substantially higher than that of AM-m (5.22 +/- 2.56 vs. 1.21 +/- 0.79 fmol/ml). In contrast, levels of AM-Gly were much lower than those of AM-m in the lung, heart, kidney, adrenal gland and liver. We also evaluated AM-Gly and AM-m levels in rats in a morbid state induced by intraperitoneal administration of lipopolysaccharide (LPS). In most tissues, levels of AM-m and AM-Gly were both increased by LPS; however, AM-Gly/AM-m ratios were not significantly affected, which suggests that AM-Gly is rapidly converted to AM-m in tissue.

Adrenal Glands↗
PubMed2003

Endothelial responses of the aorta from adrenomedullin transgenic mice and knockout mice.

Adrenomedullin (AM) is a potent vascular wall-derived vasorelaxing peptide which induces the release of nitric oxide (NO). To explore the role of endogenous AM in vascular function, we examined the effects of acetylcholine (ACh), AM, and AM receptor antagonists [AM (22-52), and calcitonin gene-related peptide (CGRP) (8-37)] on the isometric tension of aortic rings isolated from AM transgenic (TG) and knockout (KO) mice and wild type littermates (WT). ACh and AM caused a dose-dependent reduction of the isometric tension of aortic rings, but the degree of vasodilatation was smaller in TG than in KO or WT (% delta tension [10(-6) mol/l ACh]: KO -69 +/- 10%, WT -39 +/- 8%, TG -29 +/- 1%, p < 0.01). On the other hand, N(G)-nitro-L-arginine methyl ester, an NO synthase inhibitor, induced greater vasoconstriction in TG (% delta tension 10(-5)mol/l: KO +78 +/- 16%, WT +99 +/- 27%, TG +184 +/- 20%, p < 0.01), whereas E-4021, a cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase inhibitor, caused greater vasodilation in TG mice. Both AM antagonists increased tension in TG to a greater extent than in KO or WT mice (% delta tension [10(-6) mol/l CGRP (8-37)]: KO +24 +/- 5%, WT +51 +/- 6%, TG +75 +/- 7%, p < 0.01). Endothelial denudation of the aorta diminished the vasoconstriction caused by the AM antagonists. In conclusion, the amounts of AM expressed in the aortic endothelium influenced baseline NO release. AM antagonists increased vascular tone in WT as well as in TG, suggesting that endogenous AM plays a physiological role in the regulation of aortic tone.

Acetylcholine↗
PubMed2003

Adrenomedullin infusion during ischemia/reperfusion attenuates left ventricular remodeling and myocardial fibrosis in rats.

Recent studies have demonstrated that the activation of protein kinase Akt attenuates myocardial ischemia/reperfusion injury. However, it remains unknown whether adrenomedullin (AM), which is also a potent Akt activator, has cardioprotective effects after ischemia/reperfusion. In the present study, Sprague-Dawley rats were exposed to a 30-min period of ischemia induced by ligation of the left coronary artery followed by 24-h reperfusion. They were randomized to receive intravenous administration of AM (0.05 microg/kg/min) or saline for 60 min after coronary ligation. We examined the hemodynamics and myocardial apoptosis 24 h after ischemia/reperfusion. Echocardiographic measurements were performed 4 weeks after ischemia/reperfusion. Myocardial infarct size was also measured histologically. AM significantly reduced left ventricular (LV) end-diastolic pressure (17 +/- 2 to 8 +/- 2 mmHg, p < 0.05) and the number of apoptotic nuclei in myocytes (387 +/- 39 to 147 +/- 72 per field, p < 0.05). AM significantly increased LV dP/dt(max) (4,803 +/- 228 to 5,672 +/- 199 mmHg/s, p < 0.05). AM significantly increased LV fractional shortening (23 +/- 2 vs. 28 +/- 2%, p < 0.05), and significantly reduced LV diastolic dimension (7.4 +/- 0.1 to 6.9 +/- 0.1 mm, p < 0.05) and myocardial infarct size (33 +/- 2 to 20 +/- 2%, p < 0.01) 4 weeks after ischemia/reperfusion. In conclusion, AM infusion during ischemia/reperfusion attenuated the development of LV remodeling and myocardial fibrosis in rats. Based on these results, the cardioprotective effects of AM may be attributed at least partly to its anti-apoptotic effect.

Adrenomedullin↗
PubMed2003

Effects of ghrelin on growth hormone secretion from cultured adenohypophysial cells in cattle.

To clarify the direct effects of ghrelin on growth hormone (GH) release from anterior pituitary (AP) cells in cattle, GH-releasing effects of human ghrelin (hGhrelin) and rat ghrelin (rGhrelin) on bovine AP cells were compared with those of GH-releasing hormone (GHRH) in vitro. The AP cells were obtained from Holstein steers and were incubated for 2 h with the peptides after incubating in DMEM for 3 days. hGhrelin and rGhrelin significantly stimulated GH release from the cultured cells at doses from 10(-10) to 10(-7) M and from 10(-9) to 10(-7) M, respectively (P<0.05). The rates of increase in GH at 10(-10), 10(-9), 10(-8) and 10(-7) M hGhrelin were 26, 26, 59 and 100% compared with controls, respectively, and those of increase in GH at 10(-9), 10(-8) and 10(-7) M rGhrelin were 58, 74 and 106%, respectively. GHRH significantly increased GH concentrations in cultured media at a dose as low as 10(-13) M compared with the control (P<0.05). When hGhrelin (10(-8) M) and GHRH (10(-8) M) were added together, the release of GH induced by both peptides was significantly greater than that by hGhrelin alone (P<0.05), and tended to be greater than that by GHRH alone. Somatostatin (SS, 10(-7) M) significantly blunted GH release induced by hGhrelin (10(-8) M) and GHRH (10(-8) M) (P<0.05). In the presence of SS, the percent increase in GH released with hGhrelin plus GHRH was 42% and 14% greater than that by either hGhrelin or GHRH alone, respectively (P<0.05). These results show that ghrelin directly stimulates the release of GH from anterior pituitary cells, and that SS modifies ghrelin-stimulated GH release in cattle.

Animals↗
PubMed2003

Plasma levels of active form of ghrelin during oral glucose tolerance test in patients with anorexia nervosa.

OBJECTIVE: Ghrelin is an acylated peptide, whose octanoyl modification is essential for its biological activities. Previous studies demonstrated that fasting plasma ghrelin levels were high in anorectic patients, suggesting ghrelin may play an important role in the pathophysiology of anorexia nervosa. However, antibodies used in previous work to measure ghrelin concentrations in human blood do not distinguish between the active form of ghrelin (active ghrelin) and desacyl ghrelin with no biological activities. Therefore, we studied plasma levels of active ghrelin during oral glucose tolerance test (OGTT) in anorectic patients, using a radioimmunoassay (RIA) specific for active ghrelin. METHODS: Active ghrelin response to OGTT was evaluated in five female anorectic patients and seven age-matched control females. All subjects were given a 75 g/225 ml glucose solution orally after overnight fasting. For RIA of active ghrelin, 1 N hydrogen chloride was added to the samples at final concentration of 0.1 N immediately after separation of plasma. RESULTS: Plasma basal levels of active ghrelin were significantly higher in anorectic patients than in controls (52.1+/-10.5 vs 21.2+/-3.1 fmol/ml, P<0.01). They were significantly decreased during OGTT in anorectic patients and in controls, reaching a nadir of 49.0+/-7.7% and 57.3+/-4.5% of the basal levels, respectively. CONCLUSION: These results suggest that hyperghrelinemia in anorectic patients is caused at least partly by increased secretion of active ghrelin and that glucose ingestion suppresses active ghrelin release in these patients.

Anorexia Nervosa↗
PubMed2003

Increased plasma ghrelin level in lung cancer cachexia.

PURPOSE: Ghrelin, a novel growth hormone-releasing peptide,has been shown to cause a positive energy balance by stimulating food intake and inducing adiposity. We sought to investigate the pathophysiology of ghrelin in cachexia associated with lung cancer. EXPERIMENTAL DESIGN: Plasma ghrelin level was measured in 43 patients with lung cancer and 21 control subjects. Patients with lung cancer were divided into two groups: patients with cachexia (n = 21) and those without cachexia (n = 22). RESULTS: Plasma ghrelin level did not significantly differ between all patients with lung cancer and controls (157 +/- 10 versus 132 +/- 8 fmol/ml, P = 0.1). However, plasma ghrelin level was significantly higher in patients with cachexia than in those without cachexia (180 +/- 17 versus 135 +/- 10 fmol/ml, P = 0.011). Furthermore, plasma ghrelin level increased significantly in patients with decreased food intake after chemotherapy (from 136 +/- 11 fmol/ml to 170 +/- 16 fmol/ml on day 8, 179 +/- 20 fmol/ml on day 21 after start of chemotherapy), although plasma ghrelin level did not significantly change in those without decreased food intake. CONCLUSIONS: Baseline plasma ghrelin level was elevated in cachectic patients with lung cancer, and follow-up plasma ghrelin level increased in patients with anorexia after chemotherapy. Considering the positive energy effects induced by ghrelin, increased ghrelin may represent a compensatory mechanism under catabolic-anabolic imbalance in cachectic patients with lung cancer.

Aged↗
PubMed2002

A transient surge of ghrelin secretion before feeding is modified by different feeding regimens in sheep.

Ghrelin is a recently identified orexigenic hormone secreted by the stomach and has been implicated in meal-time hunger. Several experiments demonstrate a transient surge in ghrelin secretion shortly before a scheduled meal, suggesting from the involvement of cephalic mechanisms. If ghrelin secretion is stimulated by hunger in sheep, plasma levels of ghrelin should be modified by different feeding regimens that affect hunger drive. To test this hypothesis, we investigated changes in plasma ghrelin concentrations in fed Suffolk rams ad libitum and in rams either twice or four times daily. Plasma ghrelin levels increased (P<0.05) abruptly just before every feeding period in sheep fed twice and four times daily and then fell shortly after feeding. Peak levels of the pre-prandial ghrelin surge were higher (P<0.01) in animals fed twice daily than in animals fed four times daily, leading to greater (P<0.05) areas under response curves over 12h. In contrast, the plasma ghrelin levels remained relatively low and constant in sheep fed ad libitum, with no evidence of surges in plasma ghrelin levels. These results confirm that the transient surge in plasma ghrelin levels occurs just before feeding and demonstrate that this can be modified by the feeding regimen in sheep.

Animals↗
PubMed2002

The effect of pituitary adenylate cyclase activating polypeptide on cultured rat cardiocytes as a cardioprotective factor.

In the cardiovascular system, pituitary adenylate cyclase activating polypeptide (PACAP) exhibits not only vasodilation but also positive inotropic action by increasing cardiac output. Then the effect of PACAP in cultured cardiovascular cells was examined. In neonatal rat myocytes, PACAP evoked concentration-dependent increase in intracellular cyclic AMP content more potently than vasoactive intestinal polypeptide (VIP). However, in neonatal rat nonmyocytes, PACAP and VIP showed equal potency. The characterization of the subtype of PACAP/VIP receptors by RT-PCR analysis revealed that PAC1 receptor mRNA is dominantly present in the myocytes, but VPAC2 receptor mRNA is abundant in the nonmyocytes. In the myocytes, PACAP did not change the protein synthesis stimulated by endothelin or by itself. However, PACAP moderately stimulated the secretion of atrial natriuretic polypeptide (ANP). On the other hand, PACAP inhibited the protein synthesis and DNA synthesis of the nonmyocytes. These indicate that PACAP might be involved in the regulation of cardiac hypertrophy and fibrosis as a cardioprotective factor.

Animals↗
PubMed2002

Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by post-translational processing.

Ghrelin, a novel 28-amino acid peptide with an n-octanoyl modification at Ser(3), was isolated from rat stomach and found to be an endogenous ligand for the growth-hormone secretagogue receptor (GHS-R). This octanoyl modification is essential for ghrelin-induced GH release. We report here the purification and identification of human ghrelin from the stomach, as well as structural analysis of the human ghrelin gene and quantitation of changes in plasma ghrelin concentration before and after gastrectomy. Human ghrelin was purified from the stomach by gel filtration and high performance liquid chromatography, using a ghrelin-specific radioimmunoassay and an intracellular calcium influx assay on a stable cell line expressing GHS-R to test the fractions. In the course of purification, we isolated human ghrelin of the expected size, as well as several other ghrelin-derived molecules. Classified into four groups by the type of acylation observed at Ser(3); these peptides were found to be non-acylated, octanoylated (C8:0), decanoylated (C10:0), and possibly decenoylated (C10:1). All peptides found were either 27 or 28 amino acids in length, the former lacking the C-terminal Arg(28), and are derived from the same ghrelin precursor through two alternative pathways. The major active form of human ghrelin is a 28-amino acid peptide octanoylated at Ser(3), as was found for rat ghrelin. Synthetic octanoylated and decanoylated ghrelins produce intracellular calcium increases in GHS-R-expressing cells and stimulate GH release in rats to a similar degree. Both ghrelin and the ghrelin-derived molecules were found to be present in plasma as well as stomach tissue. Plasma levels of immunoreactive ghrelin after total gastrectomy in three patients were reduced to approximately half of their pre-gastrectomy values, after which they gradually increased. This suggests that the stomach is the major source of circulating ghrelin and that other tissues compensate for the loss of ghrelin production after gastrectomy.

Aged↗
PubMed2002

Chicken ghrelin and growth hormone-releasing peptide-2 inhibit food intake of neonatal chicks.

Ghrelin is an endogenous ligand for the growth hormone secretagogue (GHS) receptor. Ghrelin stimulates feeding in rats, however, intracerebroventricular (i.c.v.) injection of rat ghrelin inhibits feeding of neonatal chicks. In the present study, the effect of i.c.v. injection of different ghrelins including chicken and bullfrog ghrelin, and synthetic GH-releasing peptide (GHRP) on feeding of neonatal chicks was investigated. Chicken ghrelin strongly suppressed feeding. To compare the inhibitory effect, chicken and rat ghrelin were examined. The suppressive effect of feeding by chicken and rat ghrelin was almost identical. Bullfrog ghrelin contains a change in the acylated amino acid from Ser to Thr, strongly suppressed feeding. The i.c.v. injection of GHRP-2 (KP-102), a synthetic GHS, also inhibited feeding. These results indicate that the chicken GHS receptor is affected by several forms of GHS, and that food intake of neonatal chicks is inhibited by GHS receptor agonists.

Animals↗
PubMed2002

Immunohistochemical localization and biochemical characterization of ghrelin in the brain and stomach of the frog Rana esculenta.

Ghrelin is a 28-amino acid n-octanoylated peptide recently isolated from the rat stomach as an endogenous ligand of the growth hormone secretagogue receptor. So far, the occurrence of ghrelin has not been investigated in submammalian vertebrates. In the present work, we have studied the anatomic distribution and biochemical characterization of ghrelin-like immunoreactivity in the brain and stomach of the frog Rana esculenta by using two distinct antisera directed against rat ghrelin. In the brain, sparse ghrelin-positive cells were detected in three nuclei of the diencephalon, namely the suprachiasmatic nucleus and the posterior tuberculum in the hypothalamus, and the posterodorsal aspect of the lateral nucleus in the thalamus. A few ghrelin-immunoreactive neurons were also found in the mesencephalon, i.e., in the pretoral gray and the anterodorsal tegmental nucleus. Ghrelin-containing fibers were widely distributed in the frog brain. In particular, diffuse networks of immunoreactive processes were observed in various regions of the telencephalon, including the medial pallium, the striatum, the nucleus of the diagonal band of Broca, the nucleus accumbens, and the amygdala. In the diencephalon, the magnocellular nucleus, the suprachiasmatic nucleus, the posterior tuberculum, and the ventrolateral and lateral thalamic nuclei were moderately to densely innervated with ghrelin-containing fibers. A moderate density of positive fibers was also found in different areas of the mesencephalon such as the nucleus of the medial longitudinal fasciculus, the pretoral gray, and the tegmentum. In the stomach, a few brightly immunofluorescent cells were detected in the mucosa. The distribution pattern and morphologic characteristics of ghrelin-containing cells in the stomach suggest that they correspond to endocrine cells. Reversed-phase high performance liquid chromatography analysis of frog brain and stomach extracts, combined with RIA detection, revealed that ghrelin-immunoreactive material eluted as a single peak with a retention time slightly shorter than that of synthetic rat ghrelin. The present data provide the first evidence that a ghrelin-related peptide is present in submammalian vertebrates. The occurrence of ghrelin-containing cells in the hypothalamus and the stomach mucosa suggests that, in amphibians, ghrelin may exert both neuroendocrine and endocrine activities.

Animals↗