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Original Studies |
Departments of Biomedical Sciences and Oncology (M.P., P.C.), Anatomy, Pharmacology and Forensic Medicine (C.G., F.C., G.M.) and Internal Medicine (E.G.), University of Turin, Turin, 10125 Italy; and Europeptides (R.D.), Argenteuil 95108, France
Address correspondence and requests for reprints to: G. Muccioli, Ph.D., Division of Pharmacology, Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Via P. Giuria 13, 10125 Turin, Italy. E-mail: muccioli{at}medfarm.unito.it
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Abstract |
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Introduction |
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A specific animal and human GHS receptor has recently been cloned (9). It is encoded by a rare messenger RNA (mRNA) with a predicted open reading frame of 366 amino acids with a transmembrane topology typified by the G-protein-coupled receptor family. Receptor transcripts are expressed in the pituitary and the hypothalamus (4, 9), and their sequence shows partial homology with the neurotensin receptor and other orphan receptors, such as GPR38, GPR39, and FM-3 (10, 11).
The hypothalamus and the pituitary gland display the highest specific GHS binding in humans and in animals (6, 7, 8, 9), though a high level of specific binding has also been found in other areas of the central nervous system, such as the cerebral (but not cerebellar) cortex, hippocampus, medulla oblongata, choroid plexuses, thalamus, striatum, and substantia nigra (4, 6). The existence of GHS receptors in the brain and pituitary gland probably accounts for the endocrine and central activities of GHS (1, 2, 3). Recent evidence indicates that the distribution of the GHS receptor is not restricted to central organs, and the expression of type I GHS receptor mRNA has been demonstrated in the human pancreas (12) and in neuroendocrine tumors (13, 14). Radioiodinated peptidyl GHS are also able to label specific binding sites in the rat and human heart (15, 16, 17). There is already evidence, both in animals and in humans (16, 18, 19, 20, 21, 22, 23), that cardiac GHS receptors could mediate biological activities that are probably independent from the effect on GH secretion. Very recently, a gastric-derived peptide, named ghrelin, has been proposed as a natural ligand for GHS receptor (24). It has been shown that ghrelin has a stimulatory effect on GH secretion in the rat, but its capacity to selectively bind the GHS receptor subtype identified with radiolabeled peptidyl GHS has not yet been elucidated.
Based on the foregoing, the aims of the present study were: 1) to investigate the presence of GHS binding sites in a wide range of peripheral endocrine and nonendocrine human tissues, by radioreceptor assay with [125I]Tyr-Ala-hexarelin; and 2) to evaluate the ability of different unlabeled GHS (including ghrelin) to compete with the radioligand for binding sites in different tissues.
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Subjects and Methods |
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Hexarelin (His-D-2Me-Trp-Ala-Trp-D-Phe-Lys-NH2), Tyr-Ala-hexarelin, human ghrelin [Gly-Ser-Ser-(O n-octanoyl)-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg-NH2], and MK 0677 (N-[1(R) {[1,2-dihydro-1-methane-sulphonylspiro-(3H-indole-3, 4'-piperidin)-1'-yl]-2-(phenylmethoxy)-ethyl}-2-amino-2-methylpropanamide methane sulphonate) were supplied by Europeptides (Argenteuil, France). Human GHRH (GHRH 1–44) and SRIF (SRIF 1–14) were purchased from Bachem A. G. Feinchemikalien, Bubendorf, Switzerland. [125I]Tyr-Ala-hexarelin (SA 1800–2100 Ci/mmol) was iodinated using a lactoperoxidase method and purified by reverse-phase high-performance liquid chromatography, as previously described (6).
Tissue samples
Various autoptic and surgical human tissue specimens were included in this study. They comprised a wide range of peripheral tissues, both from endocrine (adrenal gland, ovary, pancreas, parathyroid, pituitary gland, testis, and thyroid gland) and nonendocrine organs (adipose tissue, aortic endothelium, aortic smooth muscle, carotid, colon, coronary, kidney, liver, lung, lymphnode, mammary gland, myocardium, prostate, salivary gland, skeletal muscle, skin, spleen, stomach, uterus, and vena cava). All postmortem tissues were obtained at autopsy from 10 patients [5 males, ranging in age from 24–63 yr (median age, 53 yr); and 5 females, ranging in age from 27–54 yr (median age 46 yr)] who died of trauma or neoplasms and were submitted to autopsy for diagnostic purposes, in years 1998 to 1999, in the Department of Pathology, University of Turin. Tissue removal had been given ethical approval by our hospital committee. Five samples of placenta from at-term spontaneous deliveries and 2 fragments each of adipose tissue, adrenal gland, colon, liver, lung, kidney, lymphnode, mammary gland, ovary, pituitary, prostate, salivary gland, skeletal muscle, skin, spleen, stomach, testis, thyroid, and uterus were also collected from surgical specimens received in the above Department in the same period. Of each specimen, a small (less than 1 cm3) tissue fragment (adjacent to the one fixed in formalin and used for histopathology) was immediately frozen at -30 C and stored for 1–14 months until processed for membrane preparation and binding studies. Although care was taken to collect all tissues from apparently normal organs, the absence of major pathological abnormalities was also confirmed by microscopic examination of adjacent tissue blocks of each specimen.
Binding studies
GHS binding assay with tissue membranes was performed using [125I]Tyr-Ala-hexarelin as radioligand (6, 25). The membrane fractions were prepared from frozen pieces of tissue using the method previously described for brain and pituitary gland (6). The thawed tissues were homogenized in 5–10 vol sucrose 0.3 mol/L. The homogenate was first centrifuged at 500 x g at 4 C for 10 min, and the supernatant was carefully decanted and subjected to a second centrifugation at 30,000 x g for 30 min. The resulting pellet was resuspended in ice-cold buffer (50 mmol/L Tris, 2 mmol/L EGTA, 0.03% bacitracin, titrated with HCl to pH 7.3) and immediately used to determine protein content (26) and for binding studies.
In preliminary experiments, it was found that equilibrium binding conditions for the different tissues were similar to those found for binding to human hypothalamus and pituitary gland (6). For saturation binding studies, tissue membranes (corresponding to 100 µg protein) were incubated in triplicate, at 0 C for 60 min, with increasing concentrations (0.25–20 nmol/L) of [125I]Tyr-Ala-hexarelin in a final vol of 500 µL assay buffer (50 mmol/L Tris, 2 mmol/L EGTA, 0.1% BSA, 0.03% bacitracin, titrated with HCl to pH 7.3). Parallel incubations, where 2.5 µmol/L unlabeled Tyr-Ala-hexarelin was also present, were used to determine nonspecific binding, which was subtracted from total binding to yield specific binding values. The binding reaction was terminated by adding ice-cold assay buffer followed by filtration through Whatman GF/B filters (Merck Eurolab s.r.e., Milan, Italy). Filters were rinsed three times with assay buffer, and the radioactivity remaining bound to the filters was measured by a Packard gamma counter A5003 (Packard Bioscience s.r.e., Milan, Italy). Specific binding was calculated as the difference between binding in the absence and in the presence of excess unlabeled Tyr-Ala-hexarelin and was expressed as fmol/mg protein. Precautions were taken to minimize variations in the binding of [125I]Tyr-Ala-hexarelin to tissue fractions. Thus, all binding studies related to one membrane preparation were carried out using the same batch of radiotracer. Saturation isotherms were transformed using the method of Scatchard (27), and the number of binding sites (maximal binding capacities) and the dissociation constant (Kd) for each tissue preparation were calculated with the Prism 3 program (GraphPad Software, Inc., San Diego, CA). To establish binding site specificity, increasing concentrations of various competitors (hexarelin, human ghrelin, MK 0677, GHRH, and SRIF) were tested in displacement assays with [125I]Tyr-Ala-hexarelin, and the IC50 values were calculated by iterative nonlinear curve-fitting program.
Statistical analysis
Values are expressed as median and range unless otherwise noted. In saturation and competition binding experiments, they are expressed as mean ± SEM. The number of subjects is indicated by n. Significant differences between groups were assessed by one-way ANOVA followed by the Mann-Whitney test or Duncan’s multiple-range test, depending on the experiments. P < 0.05 was chosen as the level of significance.
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Results |
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Wide variations of binding were observed among the tissues studied
(Table 1
). The highest
[125I]Tyr-Ala-hexarelin binding activity was
observed in the myocardium of both sexes and ranged from 3712–4856
fmol/mg protein. The specific binding in the myocardium represented
70–82% of the total radioactivity bound. Adrenal gland, testis,
aortic smooth muscle, aortic endothelium, coronary, carotid, lung, and
ovary possessed intermediate binding, with values ranging from
1665–3223 fmol/mg protein; liver, skeletal muscle, kidney, pituitary
gland, and thyroid gland also showed significant binding, in the range
of 689-1725 fmol/mg protein. Low binding, ranging from 108–673 fmol/mg
protein, was detected in membranes from adipose tissue, vena cava,
uterus, skin, and lymphnode. By contrast, negligible binding (<10
fmol/mg protein) was seen in the remaining tissues (parathyroid gland,
pancreas, placenta, mammary gland, prostate, salivary gland, stomach,
colon, and spleen). Differences in the specific binding values were
statistically significant when comparing the myocardium with the other
tissues (P < 0.001 in both sexes); likewise the values
obtained in adrenal gland, testis, aortic smooth muscle, aortic
endothelium, coronary, carotid, lung, and ovary differed significantly
(P < 0.01 in both sexes) from those of liver, skeletal
muscle, and kidney; differences in the binding values were also
statistically significant for liver, skeletal muscle, and kidney
vs pituitary gland and thyroid gland (P <
0.05 in both sexes) and for pituitary gland and thyroid gland
vs. adipose tissue, vena cava, skin, uterus, and lymphnode
(P < 0.001 in both sexes). No sex differences in the
Tyr-Ala-hexarelin binding to various organs were observed.
Determinations performed with the same batch of radiolabeled
Tyr-Ala-hexarelin, on membranes from surgical or autopsy specimens of
the same tissues, yielded overlapping binding values.
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. Experiments using increasing
concentrations of radiotracer revealed evidence of saturable specific
binding in the myocardium and adrenal gland (Fig. 1a). Scatchard
analysis of these data (Fig. 1b) demonstrated the existence in both
tissues of a single class of high-affinity sites with
Kd values not substantially different from one
another, being 5.5 ± 1.3 nmol/L for males (n = 5)
and 5.4 ± 1.6 nmol/L for females (n = 5) in the
myocardium and 6.5 ± 1.2 nmol/L (n = 5) for males
and 5.9 ± 1.4 nmol/L for females (n = 5) in the
adrenal gland. Kd values very close to those
found in the above tissues were also detected in the other GHS-receptor
positive tissues of both sexes.
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The specificity of [125I]Tyr-Ala-hexarelin
binding to tissue membranes was assessed by competitive binding studies
with different GHS. Table 2 shows the
IC50 values of hexarelin, MK 0677 and human
ghrelin calculated from the respective displacement curves in various
endocrine and nonendocrine organs. Unlabeled hexarelin completely
displaced radiolabeled Tyr-Ala-hexarelin from binding sites. The
concentration of hexarelin required to inhibit radiotracer binding by
50% did not significantly differ among the various tissues examined
and ranged from 2.7 to 5.5 x
10-8 mol/L. When human
ghrelin and MK 0677 were tested in the displacement studies, a
dose-dependent inhibition of binding was seen with both compounds only
in the adrenal gland (Fig. 2) and in
various endocrine organs such testis, ovary, pituitary and thyroid
gland. In these tissues human ghrelin and MK 0677 completely displaced
[125I]Tyr-Ala-hexarelin with equal efficacy,
but they were significantly (P < 0.05) less potent
than hexarelin (4–5 times). By contrast, in the myocardium (Fig. 2),
as well as in the other nonendocrine tissues, human ghrelin and MK 0677
(tested at concentrations of 0.1 nmol/L –2 µmol/L) were able to
displace only 11–31% of the specifically bound radiolabeled
Tyr-Ala-hexarelin. Other peptides (GHRH, SRIF) that have a known
influence on GH release had no effect on the binding of
[125I]Tyr-Ala-hexarelin to receptors of
endocrine and nonendocrine tissues (data not shown).
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Discussion |
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In the present study we have found that several endocrine and nonendocrine peripheral human tissues show specific binding values for Tyr-Ala-hexarelin which are even higher than found in the pituitary gland. This was found to be the case in the myocardium, adrenal, testis, arteries, lung, ovary, liver, skeletal muscle, and kidney. Specific binding values quite close to those detected in the pituitary were found in the thyroid gland. Lower levels of binding were observed, on the other hand, in adipose tissue, veins, uterus, skin and lymphnode, while negligible binding was found in parathyroid glands, pancreas, placenta, mammary gland, prostate, salivary gland, stomach, colon, and spleen. These findings indicate that GHS have widely spread receptors in some (but not all) peripheral endocrine and nonendocrine human tissues which could mediate effects other than classical endocrine and central activities (33). Evidence has been provided that treatment with peptidyl GHS exerts cardiovascular activities. In fact, coronary vasoconstriction (16) or protective effect against ischemia depending on dose and experimental conditions (18, 19, 20) and improvement of cardiac performances after myocardial infarction (23) have been observed in rats, while an increase in the left ventricular ejection fraction has been reported in humans (21). It will be noted that the myocardial effects of peptidyl GHS are elicited also in animals (18) and humans (20, 22) with severe GH deficiency. In addition, no cardiac effects were noted after giving nonpeptidyl GHS. One can therefore argue that the GHS binding sites in the heart are specific for peptidyl GHS only and mediate cardiovascular activities indepentently from their GH-releasing effect. This is not the case of the thyroid where both peptidyl and nonpeptidyl GHS displaced Tyr-Ala-hexarelin from binding sites of normal and follicular-derived neoplastic tissues and carcinoma cell lines. The thymidine incorporation and the proliferation of the latter is inhibited by both peptidyl and nonpeptidyl GHS (25). Finally, it is noteworthy that among peripheral tissues lacking GHS binding there was the stomach which has been shown as the major site of ghrelin synthesis and release (24) and the pancreas where GHS-receptor mRNA has been demonstrated (12). These data further indicate the complexity of the GHS compound/receptor interactions. Even the discovery of ghrelin as a natural GHS-like ligand (24) has not completely clarified the whole matter.
The binding of [125I]Tyr-Ala-hexarelin to membranes from peripheral human tissues showed many of the properties typical of the ligand-receptor interaction. These included high affinity, saturability and structural specificity. The specificity of radioiodinated Tyr-Ala-hexarelin binding was very similar to that observed in other human peptidyl GHS target tissues such as the hypothalamus and the pituitary gland (6), since the binding of radioligand was displaced by unlabeled Tyr-Ala-hexarelin, hexarelin, but not by peptides (GHRH or SRIF) structurally unrelated to peptidyl GHS.
The peripheral GHS binding sites do not necessarily reflect the peripheral distribution of the classic GHS receptor cloned by Howard et al. (4, 8, 9). At least in some tissues, they could even reflect the existence of GHS receptor subtypes different from that previously characterized in the pituitary (6, 7). In our study binding specificity showed remarkable differences among tissues displaying binding sites. In fact, the binding of [125I]Tyr-Ala-hexarelin was inhibited by ghrelin as well as by the nonpeptidyl spiroindoline MK 0677 in membranes from endocrine but not from nonendocrine tissues. It is worth noticing that in membranes from endocrine tissues ghrelin and MK 0677 inhibited the binding of radiolabeled Tyr-Ala-hexarelin but to a lower extent than that shown by peptidyl GHS. In all, these data clearly point toward the existence of new GHS receptor subtypes which in peripheral nonendocrine tissues seem specific for peptidyl GHS and do not bind ghrelin nor the spiroindoline MK 0677.
In conclusion, the present data demonstrate that GHS have specific receptors in a wide range of endocrine and nonendocrine human tissues and suggest that a still unknown receptor subtype, specific for peptidyl GHS, may exist in the heart and in other tissues.
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Acknowledgments |
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Footnotes |
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Received March 27, 2000.
Revised June 23, 2000.
Accepted June 30, 2000.
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 M. S. Kim, C. Y. Yoon, P. G. Jang, Y. J. Park, C. S. Shin, H. S. Park, J. W. Ryu, Y. K. Pak, J. Y. Park, K. U. Lee, et al. The Mitogenic and Antiapoptotic Actions of Ghrelin in 3T3-L1 Adipocytes Mol. Endocrinol., September 1, 2004; 18(9): 2291 - 2301. [Abstract] [Full Text] [PDF]
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R. Wu, M. Zhou, X. Cui, H. H. Simms, and P. Wang Upregulation of cardiovascular ghrelin receptor occurs in the hyperdynamic phase of sepsis Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1296 - H1302. [Abstract] [Full Text] [PDF]
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A. J. van der Lely, M. Tschop, M. L. Heiman, and E. Ghigo Biological, Physiological, Pathophysiological, and Pharmacological Aspects of Ghrelin Endocr. Rev., June 1, 2004; 25(3): 426 - 457. [Abstract] [Full Text] [PDF]
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 W. G. Li, D. Gavrila, X. Liu, L. Wang, S. Gunnlaugsson, L. L. Stoll, M. L. McCormick, C. D. Sigmund, C. Tang, and N. L. Weintraub Ghrelin Inhibits Proinflammatory Responses and Nuclear Factor-{kappa}B Activation in Human Endothelial Cells Circulation, May 11, 2004; 109(18): 2221 - 2226. [Abstract] [Full Text] [PDF]
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 Y. Sun, P. Wang, H. Zheng, and R. G. Smith Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor PNAS, March 30, 2004; 101(13): 4679 - 4684. [Abstract] [Full Text] [PDF]
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 A. INUI, A. ASAKAWA, C. Y. BOWERS, G. MANTOVANI, A. LAVIANO, M. M. MEGUID, and M. FUJIMIYA Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ FASEB J, March 1, 2004; 18(3): 439 - 456. [Abstract] [Full Text] [PDF]
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J. P. Camina, M. C. Carreira, S. El Messari, C. Llorens-Cortes, R. G. Smith, and F. F. Casanueva Desensitization and Endocytosis Mechanisms of Ghrelin-Activated Growth Hormone Secretagogue Receptor 1a Endocrinology, February 1, 2004; 145(2): 930 - 940. [Abstract] [Full Text] [PDF]
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F. Gaytan, M. L. Barreiro, J. E. Caminos, L. K. Chopin, A. C. Herington, C. Morales, L. Pinilla, R. Paniagua, M. Nistal, F. F. Casanueva, et al. Expression of Ghrelin and Its Functional Receptor, the Type 1a Growth Hormone Secretagogue Receptor, in Normal Human Testis and Testicular Tumors J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 400 - 409. [Abstract] [Full Text] [PDF]
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N. M. Thompson, D. A. S. Gill, R. Davies, N. Loveridge, P. A. Houston, I. C. A. F. Robinson, and T. Wells Ghrelin and Des-Octanoyl Ghrelin Promote Adipogenesis Directly in Vivo by a Mechanism Independent of the Type 1a Growth Hormone Secretagogue Receptor Endocrinology, January 1, 2004; 145(1): 234 - 242. [Abstract] [Full Text] [PDF]
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F. Tassone, F. Broglio, S. Destefanis, S. Rovere, A. Benso, C. Gottero, F. Prodam, R. Rossetto, C. Gauna, A. J. van der Lely, et al. Neuroendocrine and Metabolic Effects of Acute Ghrelin Administration in Human Obesity J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5478 - 5483. [Abstract] [Full Text] [PDF]
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X.-B. Xu, J.-M. Cao, J.-J. Pang, R.-K. Xu, C. Ni, W.-L. Zhu, K. Asotra, M.-C. Chen, and C. Chen The Positive Inotropic and Calcium-Mobilizing Effects of Growth Hormone-Releasing Peptides on Rat Heart Endocrinology, November 1, 2003; 144(11): 5050 - 5057. [Abstract] [Full Text] [PDF]
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U. Pagotto, A. Gambineri, C. Pelusi, S. Genghini, M. Cacciari, B. Otto, T. Castaneda, M. Tschop, and R. Pasquali Testosterone Replacement Therapy Restores Normal Ghrelin in Hypogonadal Men J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4139 - 4143. [Abstract] [Full Text] [PDF]
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F. Broglio, C. Gottero, A. Benso, F. Prodam, S. Destefanis, C. Gauna, M. Maccario, R. Deghenghi, A. J. van der Lely, and E. Ghigo Effects of Ghrelin on the Insulin and Glycemic Responses to Glucose, Arginine, or Free Fatty Acids Load in Humans J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4268 - 4272. [Abstract] [Full Text] [PDF]
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 I.A. Harsch, P.C. Konturek, C. Koebnick, P.P. Kuehnlein, F.S. Fuchs, S. Pour Schahin, G.H. Wiest, E.G. Hahn, T. Lohmann, and J.H. Ficker Leptin and ghrelin levels in patients with obstructive sleep apnoea: effect of CPAP treatment Eur. Respir. J., August 1, 2003; 22(2): 251 - 257. [Abstract] [Full Text] [PDF]
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 Y.-T. Shen, J. J. Lynch, R. J. Hargreaves, and R. J. Gould A Growth Hormone Secretagogue Prevents Ischemic-Induced Mortality Independently of the Growth Hormone Pathway in Dogs with Chronic Dilated Cardiomyopathy J. Pharmacol. Exp. Ther., August 1, 2003; 306(2): 815 - 820. [Abstract] [Full Text] [PDF]
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K. Kawamura, N. Sato, J. Fukuda, H. Kodama, J. Kumagai, H. Tanikawa, A. Nakamura, Y. Honda, T. Sato, and T. Tanaka Ghrelin Inhibits the Development of Mouse Preimplantation Embryos in Vitro Endocrinology, June 1, 2003; 144(6): 2623 - 2633. [Abstract] [Full Text] [PDF]
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K. Tanaka, H. Minoura, T. Isobe, H. Yonaha, H. Kawato, D. F. Wang, T. Yoshida, M. Kojima, K. Kangawa, and N. Toyoda Ghrelin Is Involved in the Decidualization of Human Endometrial Stromal Cells J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2335 - 2340. [Abstract] [Full Text] [PDF]
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A. Torsello, E. Bresciani, G. Rossoni, R. Avallone, G. Tulipano, D. Cocchi, I. Bulgarelli, R. Deghenghi, F. Berti, and V. Locatelli Ghrelin Plays a Minor Role in the Physiological Control of Cardiac Function in the Rat Endocrinology, May 1, 2003; 144(5): 1787 - 1792. [Abstract] [Full Text] [PDF]
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I. Depoortere, T. Thijs, L. Thielemans, P. Robberecht, and T. L. Peeters Interaction of the Growth Hormone-Releasing Peptides Ghrelin and Growth Hormone-Releasing Peptide-6 with the Motilin Receptor in the Rabbit Gastric Antrum J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 660 - 667. [Abstract] [Full Text] [PDF]
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 M.L. Barreiro, J.S. Suominen, F. Gaytan, L. Pinilla, L.K. Chopin, F.F. Casanueva, C. Dieguez, E. Aguilar, J. Toppari, and M. Tena-Sempere Developmental, Stage-Specific, and Hormonally Regulated Expression of Growth Hormone Secretagogue Receptor Messenger RNA in Rat Testis Biol Reprod, May 1, 2003; 68(5): 1631 - 1640. [Abstract] [Full Text] [PDF]
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M. Arosio, C. L. Ronchi, C. Gebbia, V. Cappiello, P. Beck-Peccoz, and M. Peracchi Stimulatory Effects of Ghrelin on Circulating Somatostatin and Pancreatic Polypeptide Levels J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 701 - 704. [Abstract] [Full Text] [PDF]
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F. Gaytan, M. L. Barreiro, L. K. Chopin, A. C. Herington, C. Morales, L. Pinilla, F. F. Casanueva, E. Aguilar, C. Dieguez, and M. Tena-Sempere Immunolocalization of Ghrelin and Its Functional Receptor, the Type 1a Growth Hormone Secretagogue Receptor, in the Cyclic Human Ovary J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 879 - 887. [Abstract] [Full Text] [PDF]
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L. M. Seoane, M. Lopez, S. Tovar, F. F. Casanueva, R. Senaris, and C. Dieguez Agouti-Related Peptide, Neuropeptide Y, and Somatostatin-Producing Neurons Are Targets for Ghrelin Actions in the Rat Hypothalamus Endocrinology, February 1, 2003; 144(2): 544 - 551. [Abstract] [Full Text] [PDF]
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 M. Volante, E. Allia, E. Fulcheri, P. Cassoni, E. Ghigo, G. Muccioli, and M. Papotti Ghrelin in Fetal Thyroid and Follicular Tumors and Cell Lines: Expression and Effects on Tumor Growth Am. J. Pathol., February 1, 2003; 162(2): 645 - 654. [Abstract] [Full Text] [PDF]
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V. Sibilia, G. Rindi, F. Pagani, D. Rapetti, V. Locatelli, A. Torsello, N. Campanini, R. Deghenghi, and C. Netti Ghrelin Protects Against Ethanol-Induced Gastric Ulcers in Rats: Studies on the Mechanisms of Action Endocrinology, January 1, 2003; 144(1): 353 - 359. [Abstract] [Full Text] [PDF]
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 G. Baldanzi, N. Filigheddu, S. Cutrupi, F. Catapano, S. Bonissoni, A. Fubini, D. Malan, G. Baj, R. Granata, F. Broglio, et al. Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT J. Cell Biol., December 23, 2002; 159(6): 1029 - 1037. [Abstract] [Full Text] [PDF]
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U. Pagotto, A. Gambineri, V. Vicennati, M. L. Heiman, M. Tschop, and R. Pasquali Plasma Ghrelin, Obesity, and the Polycystic Ovary Syndrome: Correlation with Insulin Resistance and Androgen Levels J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5625 - 5629. [Abstract] [Full Text] [PDF]
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M.L. Barreiro, F. Gaytan, J.E. Caminos, L. Pinilla, F.F. Casanueva, E. Aguilar, C. Dieguez, and M. Tena-Sempere Cellular Location and Hormonal Regulation of Ghrelin Expression in Rat Testis Biol Reprod, December 1, 2002; 67(6): 1768 - 1776. [Abstract] [Full Text] [PDF]
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H. Tamura, J. Kamegai, T. Shimizu, S. Ishii, H. Sugihara, and S. Oikawa Ghrelin Stimulates GH But Not Food Intake in Arcuate Nucleus Ablated Rats Endocrinology, September 1, 2002; 143(9): 3268 - 3275. [Abstract] [Full Text] [PDF]
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 M. Volante, E. Fulcheri, E. Allia, M. Cerrato, A. Pucci, and M. Papotti Ghrelin Expression in Fetal, Infant, and Adult Human Lung J. Histochem. Cytochem., August 1, 2002; 50(8): 1013 - 1021. [Abstract] [Full Text] [PDF]
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F. Broglio, E. Arvat, A. Benso, C. Gottero, F. Prodam, S. Grottoli, M. Papotti, G. Muccioli, A. J. van der Lely, R. Deghenghi, et al. Endocrine Activities of Cortistatin-14 and Its Interaction with GHRH and Ghrelin in Humans J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3783 - 3790. [Abstract] [Full Text] [PDF]
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 V. Bodart, M. Febbraio, A. Demers, N. McNicoll, P. Pohankova, A. Perreault, T. Sejlitz, E. Escher, R.L. Silverstein, D. Lamontagne, et al. CD36 Mediates the Cardiovascular Action of Growth Hormone-Releasing Peptides in the Heart Circ. Res., May 3, 2002; 90(8): 844 - 849. [Abstract] [Full Text] [PDF]
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M. Volante, E. AllIa, P. Gugliotta, A. Funaro, F. Broglio, R. Deghenghi, G. Muccioli, E. Ghigo, and M. Papotti Expression of Ghrelin and of the GH Secretagogue Receptor by Pancreatic Islet Cells and Related Endocrine Tumors J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1300 - 1308. [Abstract] [Full Text] [PDF]
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C. Ghe, P. Cassoni, F. Catapano, T. Marrocco, R. Deghenghi, E. Ghigo, G. Muccioli, and M. Papotti The Antiproliferative Effect of Synthetic Peptidyl GH Secretagogues in Human CALU-1 Lung Carcinoma Cells Endocrinology, February 1, 2002; 143(2): 484 - 491. [Abstract] [Full Text] [PDF]
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M. Tschop, M. A. Statnick, T. M. Suter, and M. L. Heiman GH-Releasing Peptide-2 Increases Fat Mass in Mice Lacking NPY: Indication for a Crucial Mediating Role of Hypothalamic Agouti-Related Protein Endocrinology, February 1, 2002; 143(2): 558 - 568. [Abstract] [Full Text] [PDF]
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M. Tena-Sempere, M. L. Barreiro, L. C. Gonzalez, F. Gaytan, F.-P. Zhang, J. E. Caminos, L. Pinilla, F. F. Casanueva, C. Dieguez, and E. Aguilar Novel Expression and Functional Role of Ghrelin in Rat Testis Endocrinology, February 1, 2002; 143(2): 717 - 725. [Abstract] [Full Text] [PDF]
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H.-M. Lee, G. Wang, E. W. Englander, M. Kojima, and G. H. Greeley Jr. Ghrelin, A New Gastrointestinal Endocrine Peptide that Stimulates Insulin Secretion: Enteric Distribution, Ontogeny, Influence of Endocrine, and Dietary Manipulations Endocrinology, January 1, 2002; 143(1): 185 - 190. [Abstract] [Full Text] [PDF]
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W. Zhang, M. Chen, X. Chen, B. J Segura, and M. W Mulholland Inhibition of pancreatic protein secretion by ghrelin in the rat J. Physiol., November 15, 2001; 537(1): 231 - 236. [Abstract] [Full Text] [PDF]
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N. Kanamoto, T. Akamizu, H. Hosoda, Y. Hataya, H. Ariyasu, K. Takaya, K. Hosoda, M. Saijo, K. Moriyama, A. Shimatsu, et al. Substantial Production of Ghrelin by a Human Medullary Thyroid Carcinoma Cell Line J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4984 - 4990. [Abstract] [Full Text] [PDF]
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M. Papotti, P. Cassoni, M. Volante, R. Deghenghi, G. Muccioli, and E. Ghigo Ghrelin-Producing Endocrine Tumors of the Stomach and Intestine J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 5052 - 5059. [Abstract] [Full Text] [PDF]
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C. Y. Bowers Unnatural Growth Hormone-Releasing Peptide Begets Natural Ghrelin J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1464 - 1469. [Full Text]
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P. Cassoni, M. Papotti, C. Ghè, F. Catapano, A. Sapino, A. Graziani, R. Deghenghi, T. Reissmann, E. Ghigo, and G. Muccioli Identification, Characterization, and Biological Activity of Specific Receptors for Natural (Ghrelin) and Synthetic Growth Hormone Secretagogues and Analogs in Human Breast Carcinomas and Cell Lines J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1738 - 1745. [Abstract] [Full Text]
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E. Arvat, M. Maccario, L. Di Vito, F. Broglio, A. Benso, C. Gottero, M. Papotti, G. Muccioli, C. Dieguez, F. F. Casanueva, et al. Endocrine Activities of Ghrelin, a Natural Growth Hormone Secretagogue (GHS), in Humans: Comparison and Interactions with Hexarelin, a Nonnatural Peptidyl GHS, and GH-Releasing Hormone J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1169 - 1174. [Abstract] [Full Text]
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V. Bodart, M. Febbraio, A. Demers, N. McNicoll, P. Pohankova, A. Perreault, T. Sejlitz, E. Escher, R.L. Silverstein, D. Lamontagne, et al. CD36 Mediates the Cardiovascular Action of Growth Hormone-Releasing Peptides in the Heart Circ. Res., May 3, 2002; 90(8): 844 - 849. [Abstract] [Full Text] [PDF]
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