Full text of DOI:10.1007/BF03343800

J. Endocrinol. Invest. 24: RC1-RC3, 2001
RAPID COMMUNICATION
Cortistatin, but not somatostatin, binds to growth hormone
secretagogue (GHS) receptors of human pituitary gland
R. Deghenghi*, M. Papotti**, E. Ghigo***, and G. Muccioli****
*Europeptides, Argenteuil, France; Departments of **Biomedical Sciences and Oncology, ***Internal Medicine and
****Anatomy, Pharmacology and Forensic Medicine, University of Turin, Italy
ABSTRACT. Antagonism between GH secretagogues (GHS) and somatostatin (SRIH) has been pos-
tulated and demonstrated, but SRIH does not bind to GHS receptors (GHS-R) and potent synthetic
peptidyl GHS (GHRP6, hexarelin) do not displace radiolabeled SRIH from its receptors. However,
non-natural SRIH octapeptide agonists (mainly lanreotide and vapreotide) displace 125I-Tyr-Ala-hexare-
lin from pituitary binding sites suggesting that an endogenous factor related to SRIH might exist and
interact with GHS-R. Our aims were to investigate the ability of different SRIH-like peptides such as
various SRIH fragments (SRIH 3-14, SRIH 7-14, SRIH 3-10, SRIH 7-10, SRIH 2-9) and a natural neu-
ropeptide that shows a high structural homology with SRIH such as cortistatin-14 (CST) to compete
with 125I-Tyr-Ala-hexarelin for human pituitary binding sites and to compare their binding affinity with
that of hexarelin and ghrelin, a gastric-derived peptidyl GHS that has been proposed as a natural lig-
and of GHS-R. While the binding of 125I-Tyr-Ala-hexarelin to pituitary membranes was completely
displaced by unlabelled hexarelin, ghrelin and CST, none of the SRIH fragments tested inhibited this
binding. Ghrelin and CST exhibited a similar affinity (4.6-5.4 x 10-7 mol/l) for the binding while hexare-
lin was more effective by about four orders of magnitude in displacing 125I-Tyr-Ala-hexarelin. Our da-
ta demonstrate for the first time that cortistatin, a natural peptide related to SRIH, binds to GHS-R
and suggest that this factor may play a role in modulating the activity of these receptors.
(J. Endocrinol. Invest. 24: RC1-RC3, 2001)
©2001, Editrice Kurtis
INTRODUCTION
Based on the foregoing, the aims of the present study were:
1) to investigate the ability of different SRIH-like peptides such
as various SRIH fragments (SRIH 3-14, SRIH 7-14, SRIH 3-10,
SRIH 7-10, SRIH 2-9) and a natural neuropeptide that shows
a high structural homology with SRIH such as cortistatin-14
(CST) (9) to compete with 125I-Tyr-Ala-hexarelin for binding
sites of human pituitary gland; and 2) to compare their bind-
ing affinity with that of hexarelin and ghrelin.
GH secretagogues (GHS) are synthetic, peptidyl [GH-re-
leasing peptides (GHRP6, hexarelin)] and nonpeptidyl
molecules that possess strong, dose-dependent, and re-
producible GH-releasing activity in vivo in several species
and in man (1). These substances act through specific re-
ceptors (GHS-R) which have been originally identified in
the pituitary gland and the hypothalamus. (2-4). GHS rep-
resent the synthetic counterpart of an endogenous peptide
(ghrelin) produced by the stomach (5) that potently stimu-
lates GH in men (6) by acting on GHS-R (7). Recently, we
observed that some synthetic somatostatin (SRIH) oc-
tapeptide agonists (mainly lanreotide and vapreotide), but
not SRIH, displaced 125I-Tyr-Ala-hexarelin from its recep-
tors suggesting that an endogenous factor related to SRIH
might exist and interact with the GHS-R (8).
MATERIALS AND METHODS
Chemicals
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],
CST (Pro-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-
Cys]-Lys-NH2), and different fragments of somatostatin-14
(SRIH): SRIH 3-14 (c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-
Phe-Thr-Ser-Cys]), SRIH 3-10 (H-Cys-Lys-Asn-Phe-Phe-
Trp-Lys-Thr-OH) , SRIH 7-14 (H-Phe-Trp-Lys-Thr-Phe-
Thr-Ser-Cys-OH), SRIH 2-9 (H-Gly-Cys-Lys-Asn-Phe-Phe-
Trp-Lys-OH) and SRIH 7-10 (H-Phe-Trp-Lys-Thr-OH)
were supplied by Europeptides (Argenteuil, France). 125I-
Key-words: Cortistatin, somatostatin, ghrelin, hexarelin, pituitary receptors.
Correspondence: Dr. R. Deghenghi, Europeptides, 9 Ave. du Marais, 95108
Argenteuil, France.
E-mail: deghenghi@calva.net
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Somatostatin-related peptides and GHS receptors
100
Tyr-Ala-hexarelin (SA 1800-2000 Ci/mmol) was iodinated
using a lactoperoxidase method and purified by reverse-
phase high performance liquid chromatography as previ-
ously described (2).
75
50
25
0
Tissue samples
hexarelin
ghrelin
cortistatin-14
SRIH 2-9
SRIH 3-10
SRIH 3-14
SRIH 7-14
SRIH 7-10
Pituitary glands were obtained at autopsy from 7 subjects (4
males and 3 females ranging in age from 39 to 64 years, me-
dian age 52 years) who died of trauma or neoplasms and
were submitted to autopsy for diagnostic purposes in the
Department of Pathology, University of Turin. Tissue was
removed 24-28 h after death with the approval of our hos-
pital Ethical Committee and immediately stored at -35 C
for 1-2 months until processed for membrane preparation
and binding studies. At histopathological examination, pi-
tuitary glands were all preserved from both an architec-
tural and cytological point of view.
-10
-9
-8
-7
-6
-5
-4
Competitor (log mol/l)
Binding studies
Fig. 1 - Displacement of 125I-Tyr-Ala-hexarelin from pituitary membranes of
male subject by hexarelin, ghrelin, and different peptides related to somato-
statin-14: SRIF 3-14, SRIF 3-10, SRIF 7-14, SRIF 2-9 and SRIF 7-10 and cortis-
tatin-14. Each point represents the mean SE of four separate experiments.
GHS binding assay with tissue membranes (30,000 g frac-
tion) was performed as previously described (2) using 125I-
Tyr-Ala-hexarelin as ligand. This hexarelin analog has been
reported to have in vivo the same GH-releasing potency of
hexarelin (10) and to be a reliable probe for labeling GHS
receptors in human tissues (7). Incubations in triplicate
were kept at 0 C for 60 min in 0.5 ml assay buffer (50
mmol/l Tris-HCl, 2 mmol/l EGTA, 20 mg/l bacitracin and 1
g/l BSA) containing 100 μg membrane protein and 0.5
nmol/l 125I-Tyr-Ala-hexarelin. Specific binding was defined as
the total radioligand bound minus that bound in the pres-
ence of 10 μmol/l unlabelled Tyr-Ala-hexarelin. Inhibition
of 125I-Tyr-Ala-hexarelin binding to pituitary membranes by
various competitors was obtained using 8 different con-
centrations (from 1 nmol/l to 10 μmol/l) of each substance.
The concentration of competitor required to inhibit ra-
diotracer binding by 50% (IC50) was calculated by iterative
nonlinear curve-fitting with the Prism 3 program (GraphPad
Software, Inc., San Diego, CA). In all assays, the binding re-
action was terminated by adding ice cold assay buffer fol-
lowed by filtration through Whatman GF/B filters. The ra-
dioactivity remaining bound to the filters was measured by
a Packard gamma counter.
displaced by increasing concentrations of unlabeled hexarelin,
ghrelin and CST, none of the SRIH fragments tested or SRIH
(8) inhibited the binding, of radiotracer. Ghrelin and CST ex-
hibited a similar affinity for the binding while hexarelin was
more effective (p<0.01) by about four orders of magnitude
in displacing 125I-Tyr-Ala-hexarelin. The IC50 values (mean
S.E. of four separate experiments) were (4.6 0.4)x10-7 mol/l
for ghrelin, (5.4 0.3)x10-7 mol/l for CST and (1.3 0.2)x10-7
mol/l for hexarelin. Determinations performed with another
batch of radiolabeled Tyr-Ala-hexarelin on membranes from
autoptic pituitary specimens of three female subjects, yielded
overlapping binding values (data not shown).
DISCUSSION
It has been clearly shown that synthetic peptidyl GHS such
as hexarelin act through GHS-R, which have been originally
identified in the pituitary gland and the hypothalamus (2-4).
Ghrelin, a 28 amino acid peptide isolated from the stomach
(5), is a natural ligand of GHS-R and, in fact, it has been shown
to displace 125I-Tyr-Ala-hexarelin from pituitary binding sites
(7). The existence of GHS-R subtypes has been already re-
ported (3, 4, 7) and the possibility that more than one single
ligand exists has been suggested (8, 11).
Statistical analysis
Values are expressed as mean SE. Significant differences
between groups were assesses by one-way ANOVA fol-
lowed by Duncan’s multiple-range test. P<0.05 was chosen
as the level of significance.
We have recently reported that some synthetic somato-
statin (SRIH) octapeptide agonists (mainly lanreotide and
vapreotide), but not native SRIH, displace 125I-Tyr-Ala-
hexarelin from its receptors (8). This evidence suggested
the working hypothesis that an endogenous factor related
to SRIH might exist and interact with the GHS-R, thus rep-
resenting another natural ligand of these receptors. To clar-
ify this hypothesis, we investigated the ability of different
SRIH-like peptides such as various SRIH fragments (SRIH
RESULTS
The ability of unlabeled hexarelin, ghrelin, and different pep-
tides related to SRIH such as SRIH 3-14, SRIH 3-10, SRIH
7-14, SRIH 2-9 and SRIH 7-10 and CST to compete with
125I-Tyr-Ala-hexarelin for the pituitary binding sites of male
subjects is reported in Figure 1. While the binding of
125I-Tyr-Ala-hexarelin to pituitary membranes was completely
RC2

R. Deghenghi, M. Papotti, E. Ghigo, et al.
4. Smith R.G., Palyha O.C., Feighner S.D., Tan C.P., McKee K.K.,
Hreniuk D.L., Yang L., Morriello G., Nargund R., Patchett A.A.,
Howard A.D. Growth hormone releasing substances: types and
their receptors. Horm. Res. 1999, 51 (Suppl. 3): 1-8.
3-14, SRIH7-14, SRIH 7-10, SRIH 2-9) and a putative natu-
ral neuropeptide such as CST, recently described in rat and
human brains (12), to compete with 125I-Tyr-Ala-hexarelin
binding sites of human pituitary gland comparing their bind-
ing affinity with that of hexarelin and ghrelin. The different
SRIH fragments were chosen as potential metabolites (13),
whereas CST, though it shares 11 of its 14 amino acids with
SRIH (9) and binds all five SRIH-receptor subtypes (14),
possesses several effects that do not parallel those of SRIH.
Our findings showing that CST, like hexarelin and ghrelin,
but not various fragments of native SRIH, displaces 125I-Tyr-
Ala-hexarelin from its pituitary receptors, suggest that this
factor may play a role in modulating the activity of GHS-R.
The evidence that CST, like hexarelin and ghrelin, binds to
GHS-R suggests that this peptide could represent another
endogenous GHS ligand; this hypothesis could, in turn, im-
ply that GHS-R is another specific receptor for CST. The
observed actions of CST strongly depend on the N-termi-
nal proline and the C-terminal lysine amide residue present
on its structure (9). On the other hand, some of most ac-
tive non-natural GHS such as GHRP6 and hexarelin also
have a lysine amide group in C-terminal position.
5. Kojima M., Hosada H., Data Y., Nakazato M., Matsuo H.,
Kankawa K. Ghrelin is a growth hormone-releasing acylated pep-
tide from stomach. Nature 1999, 402: 656-660.
6. Arvat E., Di Vito L., Broglio F., Papotti M., Muccioli G., Dieguez,
C., Casanueva F., Deghenghi R., Camanni F., Ghigo E. Preliminary
evidence that ghrelin, the natural GH secretagogue (GHS)-re-
ceptor ligand, strongly stimulates GH secretion in humans. J.
Endocrinol. Invest. 2000, 23: 493-495.
7. Papotti M., Ghè C., Cassoni P., Catapano F., Deghenghi R., Ghigo
E., Muccioli G. Growth hormone secretagogue binding sites in
peripheral human tissues. J. Clin. Endocrinol. Metab. 2000 (in
press).
8. Deghenghi R., Papotti M., Locatelli V., Ghigo E., Muccioli G.
Somatostatin octapeptides (lanreotide, octreotide, vapreotide
and their analogs) share the growth hormone-releasing peptide
receptor in the human pituitary gland. Third International
Symposium on Growth Hormone Secretagogues, Febr. 17-19,
2000 Keystone, CO. Endocrine 2000, in press.
This preliminary study shows for the first time a functional
link between cortistatin and GHS-R in humans, which will be
clarified in future studies on the endocrine effects of CST.
9. Criado J.R., Li H., Jiang X., Spina M., Huitron-Resendiz S., Liapakis
G., Calbet M., Siehler S., Henriksen S.J., Koob G., Hoyer D.,
Sutcliffe J.G., Goodman M., de Lecea L. Structural and composi-
tional determinants of cortistatin activity. J. Neurosci. Res. 1999,
56: 611-619.
ACKNOWLEDGMENTS
10. Arvat E., Di Vito L., Lanfranco F., Broglio F., Giordano R., Benso
A., Muccioli G., Deghenghi R., Ghigo E. Tyr-Ala-hexarelin, a syn-
thetic octapeptide, possesses the same endocrine activities of
hexarelin and GHRP2 in humans. J. Endocrinol. Invest. 1999, 22:
91-97.
The authors wish to thank Dr. I.C.A.F. Robinson for suggesting CST as
a potential ligand and Dr. C. Ghè and Dr. F. Catapano for their skilful
technical assistance. This work was supported in part by grants (ex-60%
to G.M. and E.G.) from the Ministero dell’Università e Ricerca Scientifica
e Tecnologica (MURST); by a grant from Foundation for the Study of
Endocrine and Metabolic Disease (SMEM) SMEM; and by a grant from
Europeptides.
11. Hosoda H., Kojima M., Matsuo H., Kangawa K. Purification and
characterization of rat des-Gln-14-ghrelin, a second endogenous
ligand for growth hormone secretagogue receptor. J. Biol. Chem.
2000, 275: 21995-22000.
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