Original Paper |
|
|||
|
Acta Biochim Biophys |
||||
|
doi:10.1111/j.1745-7270.2008.00457.x |
Inhibition of proliferation and apoptosis
of vascular smooth muscle cells by ghrelin
Min Zhang, Fang Yuan, Hua Liu, Hui Chen, Xingbiao Qiu, and Weiyi
Fang*
The Affiliated Chest Hospital, Shanghai
Jiaotong University, Shanghai 200030, China
Received: April 4,
2008
Accepted: June 24,
2008
This work was
supported by a grant from Science and Technology Commission of Shanghai
Municipality (No. 074119635)
*Corresponding
author: Tel, 86-21-62821990; Fax, 86-21-52306186; E-mail, [email protected]
To evaluate the possible role of ghrelin in
the development of atherosclerosis, its effects on tumor necrosis factor (TNF)-a-induced proliferation and apoptosis of
vascular smooth muscle cells (VSMCs) were investigated. Rat VSMCs were
pretreated with different concentrations of ghrelin and then with TNF-a. VSMC proliferation was measured by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and flow
cytometry method. Apoptosis was detected using propidium iodide and Annexin-V
labeling method. Exogenous ghrelin (10–1000 ng/ml) significantly inhibited TNF-a-induced proliferation of VSMCs in a
concentration-dependent manner. Treatment with 1000 ng/ml ghrelin was most
effective at inhibiting VSMC proliferation rate and the expression of
proliferating cell nuclear antigen. However, treatment with des-acyl ghrelin
affected neither proliferation nor PCNA expression. In contrast, TNF-a-induced apoptosis of VSMCs was inhibited
by both ghrelin and des-acyl ghrelin in concentration-dependent manners, with
maximal inhibition observed for both compounds at 1000 ng/ml. Taken together,
our results suggested that ghrelin inhibited both the proliferation and
apoptosis of rat VSMCs. Furthermore, the former effect is probably mediated by
the growth hormone secretagogue receptor type 1a receptor, while the latter
effect may be mediated through other receptors.
Keywords ghrelin; vascular smooth muscle
cell; atherosclerosis; proliferation; apoptosis
Ghrelin, a brain-gut peptide, was discovered in 1999. To date, it is
the only natural ligand identified for growth hormone secretagogue receptor
(GHSR) [1]. Binding to its receptor, ghrelin is able to promote the release of
growth hormone, which is involved in the regulation of energy metabolism.
Abnormal ghrelin signaling has been implicated in various diseases and
physiological processes. Ghrelin is abundantly distributed in the
cardiovascular system. A recent study demonstrated that ghrelin receptor
density was higher in the cardiovascular system of atherosclerotic mice than
in that of normal control mice, thereby suggesting ghrelin and its receptor
might play a vital role in this disease [2]. Other studies have reported that
ghrelin exhibited protective effects against the development of
atherosclerosis by increasing coronary blood flow [3], improving endothelial
function [4], inhibiting endothelial injury [5], producing vasodilation and
enhancing cholesterol efflux in macrophages [6]. Furthermore, ghrelin has been
reported to exert anti-inflammatory effects on the cardiovascular system. For
example, ghrelin was shown to inhibit both tumor necrosis factor (TNF)-a-induced
cytokine release and NF-kB activity in human umbilical vein endothelial cells (HUVECs) [7].
Furthermore, we found that ghrelin down-regulated CD40 expression in HUVECs
[8], suggesting that it might be involved in regulating the inflammatory
process of atherosclerosis. Therefore, ghrelin and its receptor appear to have
very important functions in the development of atherosclerosis.
The proliferation and apoptosis of smooth muscle cells (SMCs) are
closely related to generation, development, plaque stability, postangioplasty
restenosis and vein graft disease [9,10]. Multiple bioactive substances, such
as TNF-a, can induce phenotypic changes in SMCs, resulting in their
activation, proliferation and migration, followed by the formation of a patch
fibrous cap [11]. Many cellular factors are capable of promoting SMC
apoptosis, which ultimately results in the transformation of stable plaques to
unstable plaques [12]. It has not yet been reported whether ghrelin has the
ability to defer the progress of atherosclerosis by inhibiting the
proliferation and apoptosis of SMCs.
In the present study, cultured VSMCs were treated with exogenous
ghrelin to determine its effects on VSMC proliferation and apoptosis and to
further elucidate the role of ghrelin in the development of atherosclerosis.
Materials and Methods
Materials
Wister rats, 6–7 weeks old, 200±10 g, were purchased from the Animal Center of
Shanghai Institutes for Biological Science, Chinese Academy of Sciences
(Shanghai, China). Dulbecco’s modified Eagle’s medium was obtained from Gibco
(Gaithersburg, USA). Ghrelin and des-acyl ghrelin were obtained from Phoenix
Pharmaceuticals (Burlingame, USA). Recombinant human TNF-a,
fluorescence-activated cell sorter (FACS) permeabilizing solution and an
Annexin V-FITC apoptosis kit were purchased from BD Biosciences (San Jose,
USA). The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
was obtained from Roche (Indianapolis, USA). Dimethyl sulfoxide (DMSO) and
propidium iodide (PI) were purchased from Sigma Chemical Company (St. Louis,
USA). Anti-proliferating cell nuclear antigen (PCNA) rat monoclonal antibodies,
PC10 and immunoglobulin G (IgG) 2a, were purchased from Santa Cruz
Biotechnology (Santa Cruz, USA). Flourescein-isothiocyanate-conjugated rabbit
anti-rat IgG was obtained from DAKO (Tokyo, Japan).
Culture and treatment of rat thoracic aortic SMCs
Thoracic aortas (100–150 g) were obtained from Wistar rats and placed in Hanks solution.
The intima and extima were carefully removed. The medial smooth muscle tissues
were cut into 1 mm3 pieces and distributed equally into Carrel’s
flasks. Cells from the tissues were cultured in an incubator at 37 ºC under 5%
CO2, followed by the addition of Dulbecco’s modified Eagle’s medium
supplemented with 20% fetal calf serum and 100 U/ml penicillin. Cells reached
confluence after 2–3 weeks and were then digested with 0.125% trypsin, passaged and
cultured continuously [13]. The presence of VSMCs was confirmed by light
microscopy, electron microscopy and immunohistochemical analysis. Cells of the
3–5 generations were used in assays.
After the replacement of the complete medium with fetal bovine
serum-free RPMI 1640 medium and an overnight culture at 37 ºC under 5% CO2, VSMCs were treated with different concentrations of TNF-a (1–1000 U/ml) in
phosphate-buffered saline (PBS) for varying amounts of time (1, 3, 5, 7, 9 and
12 h). An equal amount of PBS was used in negative control experiments.
After substituting the complete medium with fetal bovine serum-free
RPMI 1640 medium, VSMCs were plated in 6-well plates and pretreated with PBS
(negative control), exogenous ghrelin (10, 100 or 1000 ng/ml) or des-acyl
ghrelin (1000 ng/ml) for 1 h prior to a 7 h treatment with 100 U/ml TNF-a at 37 ºC under
5% CO2.
Assessment of VSMC proliferation by MTT assay
MTT assays were carried out as previously described [14]. Briefly,
VSMCs obtained from rat aortas were cultured in 96-well plates in 100 ml medium at 37
ºC under 5% CO2 for 24–96 h, followed by a 4 h
incubation with 10 ml MTT (0.5 mg/ml). Next, solubilization solution (100 ml DMSO) was
added to each well and incubated overnight. Once the purple crystals had
completely dissolved, the absorbance of the samples was measured at 570 nm
using a UV/VIS spectrophotometer (u-2000; Hitachi, Tokyo, Japan).
Proliferation rate was calculated as following.
Eq.
Samples before intervention were used as blank controls.
Detection of PCNA expression by indirect immunofluorescence flow
cytometry (FCM)
VSMCs were collected from 6-well plates, placed in Eppendorf tubes, washed
with PBS, fixed with 1% phosphonoformate for 30 min, washed again with PBS and
centrifuged at 161 g for 10 min. The cells were then permeabilized with
FACS permeabilizing solution for 10 min, washed with PBS and centrifuged at 161
g for 10 min. Cells were incubated with 50 ml IgG2a for 5 h at 4 ºC and
washed three times with PBS. Then, 10 ml flourescein-isothiocyanate-conjugated IgG
was added to each tube for 30 min at 4 ºC. After the removal of unbound
antibodies by washing, the cells were resuspended in 200 ml PBS, fixed
with 1% phosphonoformate and assessed by FCM. A total of 1´104 cells were collected for analysis using
FACScan (Becton Dickson, Franklin Lakes, USA) and Cell Quest software (San
Jose, USA), and the mean fluorescence intensity (MFI) was calculated.
Analysis of VSMC DNA contents by PI staining
Apoptosis of VSMCs was analyzed by Annexin V staining and detected
as previously reported [15]. The DNA contents of VSMCs were determined by PI
staining and FACS analysis. Attached cultured VSMCs were trypsinized, mixed
with suspended cells, centrifuged at 252 g for 5 min, washed with PBS,
fixed with formaldehyde, incubated with RNase and stained with PI at a final
concentration of 25 mg/ml. FCM Cell Fit 2.02 software (Microscience, Phoenix Technology,
Washington, USA) was used to analyze the stained cells.
VSMC apoptosis detected by Annexin V staining
Cultured rat aortic VSMCs were collected, washed with PBS and
resuspended in 1´binding buffer to a final density of 1´106 cells/ml. Then 100 ml aliquots of the cell
suspension were transferred into 5 ml tubes, followed by the addition of 5 ml Annexin V-FITC
and 5 ml PI to each tube. The cells and reagents were thoroughly mixed and
incubated at 25 ºC in the dark for 15 min, and then 40 ml 1´binding buffer was added. FCM was carried out
within 1 h, and suitable negative and positive controls were included. The
percentages of Annexin V-positive cells were calculated and analyzed using Cell
Quest software [15].
Statistical analysis
Each experiment was repeated three times. SAS software (SAS
Institute, Cary, USA) was used for statistical analysis and data were
expressed as mean±SD. Comparisons between data were carried out by
single-factor analysis of variance, and differences were considered
statistically significant at P<0.05.
Results
Identified VSMCs using microscope and immunohistochemistry
VSMCs were identified using light microscopy, electron microscopy and
immunohistochemical analysis. Under light microscopy (Olympus, Tokyo, Japan),
cells appeared spindle-shaped and were arranged into bundles. Multiple layers
of cells were observed in several areas, while single layers grew in other
areas. The characteristic peak-and-valley features of VSMC colonies were also
evident [Fig. 1(A)]. Under electron microscopy (H-550; Hitachi), muscle
fibrils parallel to the long axis and associated dense bodies were evident in
the cytoplasm of the cultured VSMCs [Fig. 1(B)]. Finally,
immunohistochemical analysis using a VSMC-specific antibody revealed that over
98% of the cells were positively stained (20 fields were randomly chosen, and
100 cells in each field were evaluated) [Fig. 1(C)]. Taken together,
these three tests served to confirm the identity and purity of the cultured
VSMCs obtained from Wistar rats.
Effects of TNF-a on VSMC proliferation evaluated by MTT assay
VSMC proliferation was enhanced following TNF-a stimulation.
The peak proliferation was found at 7 h after TNF-a treatment. By 9 h after
TNF-a treatment, VSMC proliferation had decreased, and the cells began to
shrink, rounded up and became detached. Stimulation of VSMCs with TNF-a at various
concentrations (1–1000 U/ml) led to a concentration-dependent increase in VSMC
proliferation, with the maximum proliferative response at 100 U/ml. Although
cell proliferation was markedly enhanced as the TNF-a concentration increased,
the cytotoxicity was also enhanced, leading to increased numbers of dead
cells (Fig. 2).
Effects of ghrelin on TNF-a-induced VSMC proliferation
evaluated by MTT assay
Ghrelin (10–1000 ng/ml) significantly inhibited 100 U/ml TNF-a-induced VSMC
proliferation in a dose-dependent manner. Treatment with 1000 ng/ml ghrelin
decreased 100 U/ml TNF-a-induced VSMC proliferation from 0.78%±0.08% to 0.39%±0.10% (P<0.01).
In contrast, treatment with 1000 ng/ml des-acyl ghrelin had no effect on 100
U/ml TNF-a-induced VSMC proliferation (0.75±0.05%; P>0.05) (Fig.
3).
Effects of ghrelin on TNF-a-induced PCNA expression
evaluated by FCM
Exogenous ghrelin inhibited TNF-a-induced PCNA expression
in a dose-dependent manner. Treatment with 1000 ng/ml ghrelin caused the
maximum inhibition, and the MFI of PCNA decreased from 782.13±66.42 to
348.35±71.56 (P<0.01). In contrast, treatment with 1000 ng/ml
des-acyl ghrelin did not inhibit TNF-a-induced PCNA expression (MFI=817.31±105.19; P>0.05)
(Fig. 4).
Effects of ghrelin on TNF-a-induced apoptosis
evaluated by PI staining
Similar dose-dependent increases in the number and proliferation of
VSMCs were obtained at TNF-a concentrations of 10–100 U/ml. However, treatment with 1000 U/ml
TNF-a caused massive decreases in cell number and proliferation. Sub-G1 peaks were observed, suggesting extensive apoptosis of the VSMCs.
Exogenous ghrelin significantly inhibited TNF-a-induced VSMC apoptosis in
a dose-dependent manner. The proportion of apoptotic cells decreased by 33.19%
after treatment with 1000 ng/ml ghrelin. Similarly, treatment with 1000 ng/ml
des-acyl ghrelin significantly inhibited TNF-a-induced VSMC apoptosis,
with the proportion of apoptotic cells decreasing by 32.50% after treatment (Fig.
5).
Effects of ghrelin on TNF-a-induced apoptosis
evaluated by Annexin V staining
Exogenous ghrelin significantly inhibited TNF-a-induced VSMC
apoptosis in a dose-dependent manner. The proportion of apoptotic cells
decreased by 27.20% after treatment with 1000 ng/ml ghrelin. Similarly, the
proportion of apoptotic cells decreased by 26.22% in response to treatment
with 1000 ng/ml des-acyl ghrelin (Fig. 6).
Discussion
VSMC proliferation and apoptosis play critical roles in the process
of aortic stenosis development, patch stability and restenosis following coronary
interventions. In the present study, cultured rat VSMCs were selected for
experiments. MTT assays and PCNA expression levels were used to evaluate VSMC
proliferation [16]. TNF-a was added to the cell cultures to simulate the activation of VSMCs
and determine the effects of TNF-a on VSMC proliferation. VSMC proliferation was
found to occur, even in the absence of external stimulation by TNF-a. Nonetheless,
TNF-a promoted VSMC proliferation. However, at extremely high doses or
after prolonged treatment periods, TNF-a caused the cells to
shrink, round up and become detached. Massive cell death at high
concentrations and after long incubation periods was noted.
The present study provided novel evidence regarding the close
relationship between exogenous ghrelin and VSMC proliferation and apoptosis.
Proliferation of VSMC is not only important during the development of an aortic
stenosis patch, but it is also a pivotal cause of postoperative restenosis. Our
results revealed that ghrelin significantly inhibited TNF-a-induced VSMC
proliferation in a dose-dependent manner, suggesting that ghrelin may function
by inhibiting the development of aortic stenosis. It has been reported that
ghrelin, an acylated peptide with numerous activities in various organ systems,
exerts most of its known effects on the body through a highly conserved
G-protein-coupled receptor, GHSR type 1a (GHSR-1a) [17]. Also, there are other
forms of ghrelin peptides, including des-acyl ghrelin, which lacks the acyl
modification, and minor acylated ghrelin species, such as n-decanoyl ghrelin.
Des-acyl ghrelin is an isomer of ghrelin that is abundantly present in serum
but unable to unite with the GHSR-1a receptor. Therefore, it has none of the
endocrine and inflammatory effects of ghrelin [18,19]. We found that ghrelin
inhibited TNF-a-induced VSMC proliferation, whereas des-acyl ghrelin (which is
incapable of binding to GHSR-1a) had no effect on VSMC proliferation. It is
therefore possible that the inhibitory effects of ghrelin on VSMC proliferation
are dependent on GHSR-1a. Indeed, it has been reported that exogenous ghrelin
inhibits the TNF-a-induced expression of cell adhesion molecules and activation of NF-kB in HUVECs, and
that these anti-inflammatory effects of ghrelin are mediated by the GHSR-1a
receptor [7]. Moreover, we previously demonstrated that exogenous ghrelin
significantly inhibits cytokine-induced CD40 expression in HUVECs, and that
this effect of ghrelin is also mediated by GHSR-1a [8]. Taken together, these
results support the supposition that the inhibitory effects of ghrelin on VSMC
proliferation, its anti-inflammatory functions and its inhibitory effects on
CD40 expression are all mediated by GHSR-1a.
VSMC apoptosis is involved in the generation and development of
aortic stenosis and ultimately results in patch instability. In this study, PI
staining and Annexin V staining were performed to analyze the cell cycle and
degree of apoptosis in VSMCs. Both ghrelin and des-acyl ghrelin inhibited TNF-a-induced VSMC
apoptosis, suggesting an identical GHSR-1a-independent anti-apoptotic mechanism
for both ghrelin and des-acyl ghrelin. However, the observed anti-apoptotic
function may be mediated by other as yet unknown receptors. For example,
Baldanzi et al discovered that ghrelin and des-acyl ghrelin both inhibit
apoptosis of endothelial cells via extracellular signal-regulated kinase 1/2
and phosphatidylinositol-3 kinase/protein kinase B signaling pathways, which
are independent of GSHR-1a [20]. Their results identified additional pathways,
but are compatible with our present findings. In addition, Muccioli et al
reported that the anti-lipolytic effects of ghrelin are not mediated through
GSHR-1a [21]. Accordingly, ghrelin appears to have both anti-proliferative and
anti-apoptotic effects on VSMCs, and may activate different signal transduction
pathways via binding to different receptors that remain to be identified.
Although the clinical role of ghrelin in the development of coronary
artery disease requires further research, its regulation of VSMC proliferation
and apoptosis may have significant theoretical and clinically applicable value
in the development of coronary aortic stenosis patches, patch stability and
postoperative restenosis. In the course of coronary aortic stenosis and restenosis,
inflammatory mediators, including cytokines, can either promote VSMC
proliferation or induce apoptosis by modulating intracellular inflammatory
responses. Ghrelin may inhibit VSMC proliferation and apoptosis by binding to
different receptors to activate diverse signaling pathways that can ultimately
defer or hinder the progress of coronary aortic stenosis and prevent
restenosis.
Taken together, the results of the present study demonstrate that
ghrelin mediates GHSR-1a-dependent inhibition of VSMC proliferation and
GHSR-1a-independent inhibition of VSMC apoptosis. These effects of ghrelin on
VSMCs may represent one mechanism by which ghrelin interferes with the
development of aortic stenosis. Although this pilot study only examined the
influence of ghrelin on VSMC proliferation and apoptosis in cell culture
settings, it lays a solid foundation for future studies. For example, the
specific parts of the signaling pathways involved in VSMC proliferation and
apoptosis that are affected by ghrelin need to be addressed and characterized.
Further research in this field is clearly warranted.
Acknowledgements
The authors thank Drs. Guoping Lu and Chunfang Wu (Department of
Cardiology, Shanghai Ruijin Hospital, Shanghai, China) for their generous help
and skillful assistance.
References
1 Kojima M, Hosoda H, Date
Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing
acylated peptide from stomach. Nature 1999, 402: 656–660
2 Katugampola SD, Maguire JJ,
Kuc RE, Wiley KE, Davenport AP. Discovery of recently adopted orphan receptors
for apelin, urotensin II, and ghrelin identified using novel radioligands and
functional role in the human cardiovascular system. Can J Physiol Pharmacol
2002, 80: 369–374
3 Chang L, Ren Y, Liu X, Li
WG, Yang J, Geng B, Weintraub NL et al. Protective effects of ghrelin on
ischemia/reperfusion injury in the isolated rat heart. J Cardiovasc
Pharmacol 2004, 43: 165–170
4 Shimizu Y, Nagaya N,
Teranishi Y, Imazu M, Yamamoto H, Shokawa T, Kangawa K et al. Ghrelin
improves endothelial dysfunction through growth hormone-independent mechanisms
in rats. Biochem Biophys Res Commun 2003, 310: 830–835
5 Li GZ, Jiang W, Zhao J,
Pan CS, Cao J, Tang CS, Chang L. Ghrelin blunted vascular calcification in
vivo and in vitro in rats. Regul Pept 2005, 129: 167–176
6 Avallone R, Demers A,
Rodrigue-Way A, Bujold K, Harb D, Anghel S, Wahli W et al. A growth
hormone-releasing peptide that binds scavenger receptor CD36 and ghrelin
receptor up-regulates sterol transporters and cholesterol efflux in macrophages
through a peroxisome proliferator-activated receptor g-dependent pathway.
Mol Endocrinol 2006, 20: 3165–3178
7 Li WG, Gavrila D, Liu X,
Wang L, Gunnlaugsson S, Stoll LL, McCormick ML et al. Ghrelin inhibits
proinflammatory responses and nuclear factor-kB activation in
human endothelial cells. Circulation 2004, 109: 2221–2226
8 Zhang M, Yuan F, Chen H,
Qiu X, Fang W. Effect of exogenous ghrelin on cell differentiation antigen 40
expression in endothelial cells. Acta Biochim Biophys Sin 2007, 39: 974–981
9 Lavezzi AM, Ottaviani G,
Matturri L. Biology of the smooth muscle cells in human atherosclerosis. APMIS
2005, 113: 112–121
10 Deng DX, Spin JM, Tsalenko A,
Vailaya A, Ben-Dor A, Yakhini Z, Tsao P et al. Molecular signatures
determining coronary artery and saphenous vein smooth muscle cell phenotypes:
distinct responses to stimuli. Arterioscler Thromb Vasc Biol 2006, 26: 1058–1065
11 Bochaton-Piallat ML, Gabbiani G.
Modulation of smooth muscle cell proliferation and migration: role of smooth
muscle cell heterogeneity. Handb Exp Pharmacol 2005, 170: 645–663
12 Niemann-Jonsson A, Ares MP, Yan
ZQ, Bu DX, Fredrikson GN, Branen L, Porn-Ares I et al. Increased rate of
apoptosis in intimal arterial smooth muscle cells through endogenous activation
of TNF receptors. Arterioscler Thromb Vasc Biol 2001, 21: 1909–1914
13 Yan Z, Hansson GK.
Overexpression of inducible nitric oxide synthase by neointimal smooth muscle
cells. Circ Res 1998, 82: 21–29
14 Zhang YK, Shen GF, Ru BG.
Survival of human metallothionein-2 transplastomic Chlamydomonas reinhardtii
to ultraviolet B exposure. Acta Biochim Biophys Sin 2006, 38: 187–193
15 Ji MJ, Su C, Wang Y, Wu HW, Cai
XP, Li GF, Zhu X et al. Characterization of CD4+ T cell responses
in mice infected with Schistosoma japonicum. Acta Biochim Biophys Sin
2006, 38: 327–334
16 Pellicciari C, Danova M,
Giordano M, Fuhrman Conti AM, Mazzini G, Wang E, Ronchetti E et al.
Expression of cell cycle related proteins—proliferating cell nuclear antigen
(PCNA) and statin—during adaptation and de-adaptation of EUE cells to a
hypertonic medium. Cell Prolif 1991, 24: 469–479
17 Young Cruz CR, Smith RG. The
growth hormone secretagogue receptor. Vitam Horm 2008, 77: 47–88
18 Hosoda H, Kojima M, Matsuo H, Kangawa K.
Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in
gastrointestinal tissue. Biochem Biophys Res Commun 2000, 279: 909–913
19 Nishi Y, Hiejima H,
Mifune H, Sato T, Kangawa K, Kojima M. Developmental changes in the pattern of
ghrelin’s acyl modification and the levels of acyl-modified ghrelin in murine
stomach. Endocrinology 2005, 146: 2709–2715
20 Baldanzi G,
Filigheddu N, Cutrupi S, Catapano F, Bonissoni S, Fubini A, Malan D et al.
Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and
endothelial cells through ERK1/2 and PI3K-AKT. J Cell Biol 2002, 159: 1029–1037
20 Muccioli G, Pons
N, Ghe C, Catapano F, Granata R, Ghigo E. Ghrelin and des-acyl ghrelin both
inhibit isoproterenol-induced lipolysis in rat adipocytes via a non-type 1a
growth hormone secretagogue receptor. Eur J Pharmacol 2004, 498: 27–35