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Introduction
Ghrelin, a novel 28-amino acid peptide principally released from the stomach, has been identified as an endogenous
ligand for the growth hormone secretagogue receptor
(GHSR)[1]. GHSR-1a, a G protein-coupled receptor mainly expressed in the
pituitary and hypothalamus, is thought to mediate the endocrine actions of ghrelin, including its potent growth hormone
(GH)-secreting effects[2]. Ghrelin mainly circulates in 2 different forms: octanoyl and des-octanoyl ghrelin.
Although ghrelin is essentially a gastro-derived hormone, it has been found to be expressed ubiquitously and circulated in
plasma at a concentration of approximately 100 pmol/L in healthy
humans[3]. Recently, growing evidence has indicated that
cardiovascular tissue is rich in ghrelin receptors, and that its mRNA is expressed abundantly in human myocardial cells, and
rat ventricles, atria, aorta, coronary arteries, carotid arteries, the endocardium and vena
cava[4], which confirms that the cardiovascular system is a target for ghrelin. In addition, ghrelin is an important autocrine/paracrine factor of cardiovascular
tissues and has various cardiovascular effects, including increased myocardial contractility and vasodilatation, and
anti-inflammation[5,6]. Our previous work showed that ghrelin was an endogenous cardioprotective factor in isolated rat hearts
with ischemia/reperfusion injury, ghrelin perfusion enhanced coronary flow, ameliorated cardiac function suppression and
decreased myocardial oxygen
consumption[7]; in rats with heart failure induced by sepsis, ghrelin improved cardiac function
and hemodyna-mics[8]; and in rats with acute myocardial necrosis induced by isoproterenol (ISO; 40 mg/kg per d, for 2 d)
ghrelin administration significantly protected the heart from ischemic injury and lipid peroxidation
damage[9]. However, the cardiopro-tective mechanism of ghrelin is still unclear.
From past studies, the effect of ghrelin was thought to depend entirely on its acylation and subsequent interaction with
GHSR to promote the synthesis and secretion of GH. Recent investigations have revealed that besides having a stimulatory
effect on GH secretion, ghrelin possesses a variety of effects through GH-independent mechanisms. Tsubota and coworkers
reported that the microinjection of ghrelin into rat nucleus tractus solitarii (NTS) elicited hypotensive effects, but this was not
the case upon injection into GHSR-expressing rostral ventrolateral medulla or caudal ventrolateral medulla. To elucidate the
mechanism of ghrelin, they examined the cardiovascular response of rats microinjected with des-acyl ghrelin into NTS.
Des-acyl ghrelin does not activate GHSR-1a and is devoid of endocrine activity. The hypotensive and bradycardic activity
evoked by des-acyl ghrelin was not significantly different from that of native ghrelin. These results suggest that des-acyl
ghrelin contri-butes to the regulation of cardiovascular control and that a receptor other than GHSR-1a exists in NTS. In
addition, several reports have shown that ghrelin plays many roles that are independent of GH
release[10-12]. However, it is unknown whether the cardioprotective role of ghrelin is absolutely or partly dependent on GH
release.
In the present study, we used an ISO-induced myocardial injury model to observe the changes in expression of cardiac
ghrelin and its receptor, and to investigate the therapeutic effects of ghrelin and des-octanoyl ghrelin and the mechanisms
thereof.
Materials and methods
Animals and reagents All animal care and experimental protocols were complied with the Animal Management Rules of
the Ministry of Health of the People¡¯s Republic of China (document
No 55, 2001) and the guidelines for the Care and Use of
Laboratory Animals of the First Hospital, Peking University. Male Sprague-Dawley (SD) rats (250-300 g) were provided by
the Animal Department of the Health Science Center of Peking University. All animals were maintained on normal rat chow,
had free access to water, and were kept in conditions of 12 h light/12 h dark cycle. Synthetic human ghrelin, des-octanoyl
ghrelin, [3H]ghrelin (54 mCi/mmol) and radioimmunoassay kits for rat ghrelin were provided by Phoenix Pharmaceuticals (St
Joseph, MO, USA). The radioimmunoassay kit for rat GH was provided by Eiken Chemicals (Tokyo, Japan). ISO and
aprotinin were purchased from Sigma (St Louis, MO, USA), and the kit for hydroxyproline was from Jiancheng Biotechnology
Institute (Nanjing, China). Trizol was from Gibco (Rockville, MD, USA), and dNTP, MuLV reverse transcriptase,
Oligo(dT)15, and Taq DNA polymerase were from Promega (Madison, WI, USA). Oligonucleotide primer sequences were as follows:
ghrelin-S, 5¡¯-CTT CTT GAG CCC AGA GCA C-3¡¯ and ghrelin-A, 5¡¯-GTG GCT GCA GTT TAG CTG GT-3¡¯, used for the
amplification of ghrelin cDNA; GHSR-S, 5¡¯-CTA TCC AGC ATG GCC TTC TC-3¡¯ and GHSR-A, 5¡¯-GGA AGC AGA TGG CGA AGT
AG-3¡¯, used for the amplification of GHSR cDNA; and beta-actin-S, 5¡¯-ATC TGG ACC ACC TTC-3¡¯, and beta-actin-A, 5¡¯-AGC CAG
GTC CAG ACG CA-3¡¯, used for the amplification of b-actin for calibrating sample loading. All sequences of oligonucleotide
primers were synthesized by Sai Bai Sheng (Beijing, China). Other chemicals and reagents were of analytical grade.
Preparation of animal model The ISO-induced subacute myocardial injury model was produced as described
previously[13], with minor modifications. Thirty male SD rats were randomly and equally divided into 5 groups: (1) ISO group: ISO (20,
10, and 5 mg/kg) was subcutaneously injected on d 1, 2, and 3, respectively, and then 3 mg/kg for the next 7 d; (2) control
group: rats were subcutaneously injected with normal saline for 10 d; (3) and (4) therapeutic groups: ISO administration was
the same as in (1), and ghrelin and des-octanoyl ghrelin (100 µg/kg, twice daily), respectively, were subcutaneously injected
for 10 d.
At the end of the experiment all rats were fasted overnight but had free access to water at the last administration of the
drug. The rats were anesthetized with urethane
(1 g/kg, ip), and a catheter filled with heparin saline (500
U/mL) was inserted into the right common carotid artery for measuring intraventricular pressure. The heart rate, maximal
left-ventricular pressure development (LV
dp/dtmax) and left ventricular end-diastolic pressure (LVEDP) were recorded by using
Powerlab/4S (Santa Monica, CA, USA). After the hemodynamic parameters were measured, a blood sample was drawn from
the left ventricle and mixed with 1 mg/mL ethylenediamine tetraacetic acid (EDTA)-2Na and 500 kIU/mL aprotinin. Serum was
obtained by centrifugation at 600×g for 10 min at 4
oC and stored at -70 oC. Hearts were removed and weighed. The heart apex
was taken for pathological examination under hematoxylin-eosin staining. The left ventricular (LV) myocardial sample (100
mg) was acidified by adding 1 mmol/L acetic acid, boiling for 10 min and homogenizing. The tissue supernatant was stored
at -70 oC and used for ghrelin radioimmunoassay.
Plasma lactate dehydrogenase (LDH) activity was measured on an automatic biochemistry analyzer. The content of lipid
peroxidation product malondialdehyde (MDA) and conjugated diene (CD) in myocardia and plasma was determined by using
the thiobarbituric acid test[14] and
spectrometry[15], respectively, in duplication. The collagen content in the LV myocardium
was determined by hydroxyproline assay as described
previously[16].
Assay of mRNA levels of ghrelin and GHSR by RT-
PCR[17] Total RNA from the LV myocardium (approximately 50 mg) was extracted by using Trizol reagent. A total of 1 µg of
total RNA was reverse-transcribed into single-strand cDNA with oligo(dT) 15 primer and M-MuLV reverse transcriptase.
RT-PCR was performed in a 0.2 mL tube containing 2 µL tissue cDNA, 1 µL of 5 µmol/L solutions of each S and A primer, 1 µL of
2.5 mmol/L of each dNTP mixture,
1.5 µL of 1.5 mmol/L MgCl2, 2.5 µL 10× PCR buffer, and 1.25 U
Taq DNA polymerase, in a total volume of 25 µL. RT-PCR of
ghrelin was as follows: after being denatured at 95
oC for 5 min, the solution underwent PCR at 94
oC for 30 s, 55 oC for 30 s, and
72 oC for 40 s for 30 cycles. RT-PCR of GHSR was as follows: after being denatured at 95
oC for 5 min, the solution underwent
PCR at 94 oC for 30 s, 58 oC for 30 s, and 72
oC for 40 s for 30 cycles. The 6 µL PCR product was separated on a 1.5% agarose
gel and stained with ethidium bromide. The optical density of the band of ghrelin mRNA (374 bp) and GHSR mRNA (321 bp)
was measured by use of the Gel Documentation System (Bio-Rad, Hercules, CA, USA). The PCR products were amplified
again at 94 oC for 30 s, 55
oC for 30 s, and 72 oC for 30 s for 20 cycles with the rat
b-actin primers
b-actin-S and b-actin-A. The optical density of the
b-actin mRNA band (291 bp) was measured. The ratio of ghrelin and GHSR
mRNA to b-actin mRNA was considered the relative amount of ghrelin and GHSR mRNA, respectively.
Radioimmunoassay of ghrelin and GH Plasma and the supernatant extracted from myocardia were loaded onto a Sep-Pak
C18 cartridge after equilibration with normal saline. The cartridge was washed with 2.5 mL normal saline and 10% acetonitrile
in 0.1% trifluoroacetic acid, then eluted with 2 mL 50% acetonitrile in 0.1% trifluoroacetic acid. The elution was lyophilized
and subjected to radioimmunoassay for ghrelin and GH determination. The
IC50 for ghrelin was 6.95 pmol/tube and the
reactivity with rat ghrelin was 100%. No cross-reactivity was seen with leptin, orexin A and B, neuropeptide Y, galanin, or
vasoactive intestinal polypeptide. The lowest sensitivity of GH was 0.1 ng/mL and the reactivity with rat GH was 100%.
[3H]Ghrelin binding to myocardial sarcolemmal membrane
Crude myocardial sarcolemmal membrane was prepared as
previously described[18] with minor modifications, and protein content was determined by using the Bradford
method[19]. The activity of membrane marker enzymes was measured as described previously by our
laboratory[20]. Marker enzyme activity of
membrane fractions isolated from control hearts and ISO-treated hearts showed the sarcolemmal membrane
Na+-K+-ATPase activity (6.50±0.60 mmol/g protein) to be 5.8-fold higher than that of the homogenate of myocardia (0.96±0.10 mmol/g protein).
The ghrelin-receptor binding assay was carried out using
[3H]ghrelin as a radioligand. The standard assay mixture
contained 10 mmol/L MgCl2, 50 mmol/L Tris-HCl (pH 7.4), and
[3H]ghrelin (1-40 nmol/L) in the presence or absence of
unlabeled ghrelin (20 µmol/L) in a final volume of 0.2 mL. The assay mixture was preincubated at 37
oC for 2 min, and the binding assay was initiated by the addition of sarcolemmal membrane (50 µg protein) and allowed to proceed for 60 min at 25
oC. At the end of incubation, the reaction mixture was diluted with 4 mL ice-cold washing buffer (10 mmol/L
MgCl2, 50 mmol/L Tris-HCl, pH 7.4) and filtered immediately on Millipore membranes through 0.45-µm glass-fiber filter paper (Baxter Healthcare,
Ireland) under suction. The filter paper was washed 3 times with 5 mL washing buffer (20
mmol/L Tris, 2 mmol/L MgCl2, pH 7.4) and dried, and the radioactivity was determined by using a multi-purpose scintillation counter (LS6500; Beckman, CA,
USA). The specific binding was defined as the bound
radioactivity displaceable by 20 µmol/L of ghrelin. All binding experiments were performed in duplicate. The maximal binding
capacity (Bmax) and the affinity [the reciprocal of the dissociation constant
(Kd)] for
[3H]ghrelin were calculated from Scatchard plot analysis results.
Morphological analysis Hearts were isolated and the cardiac apex was fixed in 10% formalin in phosphate-buffered saline
(PBS). Tissue was embedded in paraffin, and transverse sections were cut and stained with hematoxylin and eosin or 0.1%
picrosirius red to stain collagen.
Statistical analysis Data are expressed as mean±SD. Comparisons between more than 2 groups were carried out by using
analysis of variance (one-way ANOVA), then the Student-Newman-Keuls test. Linear regression analysis was used to
assess the correlation between variables.
P<0.05 was considered statistically significant.
Results
ISO induced myocardial ischemia injury and cardiac fibrosis
The hearts of rats treated with ISO alone became markedly
enlarged, and the heart weight and the heart coefficient (ratio of heart weight to body weight) were increased by 42% and
58%, respectively (all P<0.01) as compared with the control group. Compared with control rats, ISO-treated rats showed
lower +LV dp/dtmax and -LV
dp/dtmax values, decreased by 66% and 65%, respectively
(P<0.01) and higher LVEDP, by 2.4-fold
(P<0.01; Table 1). LDH activity in plasma was greatly increased, by 3.9-fold
(P<0.01); the MDA and CD content was elevated
in myocardia, by 74% and 69%, respectively
(P<0.01), and in plasma, by 180% and 52%, respectively
(P<0.01; Table 2). Histological sections of ISO-treated hearts showed widespread subendocardial necrosis, muscle fibers with capillary dilatation,
leukocytic infiltration, swelled fibroblasts (Figure 1A), and abundant fibroblastic hyperplasia (Figure 1B).
Treatment with ISO increased myocardial ghrelin content and mRNA level
Compared with the control group, ISO-treated rats showed increased irradiated-ghrelin (ir-ghrelin) content in plasma by 67% (246±17
vs 410±32 nmol/L, P<0.01) and
myocardia by 66% (10.41±0.57 vs 17.24+1.38 fmol/mg protein,
P<0.01), and increased mRNA level in myocardia, by 93%
(P<0.01; Figure 2). The ir-ghrelin content in myocardia and plasma was negatively correlated with +LV
dp/dtmax (r=
-0.713 and r=-0.672, respectively, both
P<0.01) and -LV
dp/dtmax (r=-0.668 and
r=-0.584, respectively, both P<0.01); and
positively correlated with LVEDP (r=0.845,
P<0.01) and (r=
0.864, P<0.01, respectively) but not hydroxyproline content
(r=0.338 and r=0.284, respectively, both
P>0.2).
Treatment with ISO increased mRNA level of myocardial GHSR and its binding capacity
Compared with the control group, ISO-treated rats showed higher GHSR mRNA levels in the myocardia, increased by 95%
(P<0.01; Figure 2). [3H]Ghrelin
binding to cardiac sarcolemmal membranes was found to be a saturable process, with single-component binding being
characteristic of all experimental groups. In the sarcolemmal membrane, the
Bmax, calculated from the results of a Scatchard
plot (Figure 3), was increased by 87% in the ISO group as compared with controls (34.38±1.66
vs 18.30±
1.64 fmol/mg protein, P<0.01). The affinity
(Kd) for [3H]ghrelin binding in sarcolemmal membranes (Figure 3) was not
significantly altered (4.31±0.42 vs 4.75±0.51 nmol/L,
P>0.05).
Administration of ghrelin or des-octanoyl ghrelin attenuated ISO-induced myocardial injury
Compared with the ISO-alone group, rats given ghrelin showed decreased heart weight and heart coefficient by 14% and 21% (all
P<
0.01), respectively; +LV
dp/dtmax and -LV
dp/dtmax were higher, increased by 1.4- and 1.2-fold
(P<0.01), respectively; and LVEDP was lower, decreased by 34%
(P<0.01, Table 1). Myocyte necrosis, as indicated by myocardial LDH leakage, was
ameliorated (P<0.01); and lipid peroxidation product, MDA and CD content in myocardia was obviously reduced, by 36% and
30% (P<0.01), respectively, and in plasma by 27% and 20% (P<0.01), respectively. Myocardial fibrosis, shown by
hydroxyproline level, was decreased by 40%
(P<0.05;
Table 2). Histological sections showed that the ISO-induced myocardial injury was obviously ameliorated by ghrelin therapy,
with the area stained by collagen markedly decreased (Figure 1A, 1B).
Des-octanoyl ghrelin administration also ameliorated the ISO-induced myocardial injury. Compared with the ISO-alone
group, ISO-treated rats showed lower heart weight and heart coefficient, decreased by 8% and 9%, respectively
(P<0.01); higher +LV
dp/dtmax and -LV
dp/dtmax values, increased by 54% and 70%
(P<0.01), respectively, and lower LVEDP, by 19%
(P<0.01; Table 1). Myocyte damage, such as myocardial LDH leakage, was ameliorated
(P<0.01), and MDA and CD content in myocardia was reduced, all by 12%
(P<0.01). MDA and CD level in plasma was decreased by 19% and 17%
(P<0.01), respectively. Fibrosis was relieved in myo-cardia, as shown by an 18% lower hydroxyproline level
(P<0.05; Table 2). Histological sections showed ameliorated ISO-induced myocardial injury and collagen deposition (Figure
1A, 1B).
The cardioprotective effects of exogenous des-octanoyl ghrelin administration on ISO-induced myocardial injury, cardiac
function suppression, cardiomegaly, collagen deposition and fibrosis were markedly weaker than those of equal doses of
ghrelin (P<0.05 or P<0.01; Table 2, Figure
1).
Effect of ghrelin and des-octanoyl ghrelin on plasma GH level
Compared with the control group, rats treated with ISO
alone showed unaltered plasma GH levels (2.88±0.31
vs
2.82±0.22 ng/mL, P>0.05); ghrelin injection greatly increased the GH level, by 1.5-fold
(P<0.01), but des-octanoyl ghrelin did not affect the plasma GH level
(P>0.05; Figure 4).
Discussion
Ghrelin is a strong gastrokinetic agent that links the endocrine control of energy balance and growth with the regulation
of digestive function by activating its specific receptor (GHSR-1a) in the central tissues. Binding sites specific for ghrelin and
its mRNA expression exist in cardiovascular tissues, which indicates that the cardiovascular system expresses the
components of the ghrelin/GHSR axis, which may have an important autocrine/paracrine function in maintaining circulatory
homeostasis. Intravenous injection of ghrelin has been shown to elicit dose-related decreases in arterial pressure and heart
rate in conscious rabbits[21]. Treatment with ghrelin causing a direct relaxation in rat thoracic aortic
rings[22] and human internal mammary
arteries[23] indicated that ghrelin is a vasorelaxing factor. Enomoto and
colleagues[24] showed that subcutaneous administration of 3 doses of ghrelin (1, 5 or 10 µg/kg) caused a dose-dependent increase in the LV ejection fraction
in healthy volunteers, which was still apparent 60 min after ghrelin injection; in patients with chronic heart failure, an
intravenous infusion of human ghrelin (0.1 mg/kg per min) significantly increased cardiac and stroke volume
index[25]. These results suggest that ghrelin could be an endogenous factor with potent protective effects against cardiovascular disease.
Over-release of catecholamines is an important factor related to myocardial impairment in many cardiovascular diseases,
such as myocardial ischemia, hypertrophy and heart failure. We found that treatment with high doses of ISO for 10 d resulted
in severe myocardial injury, including cardio-megaly, infarction-like necrosis and massive fibrosis, as did Rona
et al[13]. Cardiac function was severely inhibited, lowering the values of ±LV
dp/dtmax and elevating the LVEDP. The increase in LDH
activity in plasma indicated the leakage of myocardial intracellular enzymes, and the increased MDA and CD content in
myocardia and plasma indicated excessive products from lipid peroxidation. The increased hydroxyproline content in
myocardia and picrosirius red-stained myocardial sections indicated myocardial fibrosis. In ISO-treated rats, the mRNA levels of
ghrelin and GHSR in myocardia were elevated, the
[3H]ghrelin Bmax of the sarcolemmal membrane was increased, and the
ir-ghrelin content in plasma and myocardia was increased, with a negative correlation between ghrelin content and cardiac
function. Furthermore, exogenous administration of ghrelin effectively ameliorated the ISO-induced myocardial injury,
cardiac function inhibition, cardiomegaly, collagen deposition and fibrosis. These results indicate a role for ghrelin as an
endogenous cardioprotective factor.
Various agents affecting endocrine and metabolism participate in the regulation of expression, synthesis and secretion of
ghrelin. Food intake, obesity and positive energy balance downregulate ghrelin level, whereas starvation and nutritional
disturbance upregulate ghrelin level; insulin, growth hormone release inhibiting hormone and gastrone restrain the
expression of ghrelin. However, little is known about how ghrelin and GHSR are regulated when produced and secreted in local
cardiovascular tissues. In both rats and humans, the ghrelin gene, located at 3p26-p25, is made up of 4 exons and 3 introns,
and the precursors contain 117 amino acids (preproghrelin). Kishimoto and
colleagures[26] cloned the human ghrelin gene
and characterized the 5V-flanking region, from -2000 to -1 upstream of the translation start site. The gene contained a
TATATAA element and putative binding sites for several transcription factors, but not a typical GC or CAAT box. Glucagon
and its second messenger cAMP enhanced the promoter activity. From our work, the upregulated myocardial ghrelin and its
receptor during ISO-induced myocardial injury needs further study.
Ghrelin is a multifunctional peptide, and is crucial in regulating energy homeostasis. It induces positive energy balance
by stimulating food intake, while decreasing fat utilization. In cardiovascular tissue, ghrelin has direct vaso-dilator and
inotropic effects[22,23]. Ghrelin increases endothelial nitric oxide synthase (eNOS) expression and nitric
oxide production[24], and interferes with endothelin-1 synthesis and
release[9]. Our present work showed that treatment with
ghrelin or des-octanoyl ghrelin ameliorated ISO-induced myocardial injury, and the protective effects of ghrelin were at least
in part mediated by its hemodynamic potency.
It is well known that oxidative stress injury and inflammatory reactions are the main mechanisms of high-dosage
ISO-induced myocardial injury. Li and
colleagues[27] confirmed that ghrelin inhibited proinflammatory cytokine production,
mononuclear cell binding, and nuclear factor-kB activation in human endothelial cells
in vitro and endotoxin-induced cytokine production
in vivo. In ISO-induced myocardial injury and in ischemia/reperfusion-induced injury in isolated rat hearts,
Chang and coworkers[9] found that ghrelin effectively scavenged oxygen free radicals and inhibited lipid peroxidation injury.
In our work, ghrelin and des-octanoyl ghrelin diminished leukocytic infiltration and the generation of MDA and CD, which
showed that the anti-inflammatory and anti-lipid peroxidation potency of ghrelin is involved in heart protection.
Ghrelin is the only endogenous ligand of GHSR, and ghrelin-activated GHSR-1a in the pituitary gland and hypothalamus
induces GH release. Many reports have found that GH administration increases cardiac
performance[28],
deflates infarct size and reduces unfavorable remodeling.
All these effects strongly slow the invasion of heart
failure[29]. Imanishi and
coworkers[30] reported that GH had an anti-
fibrotic effect on cardiac remodeling through a downregula-tion of tumor growth
factor-b (TGF-b) signaling via dephosphorylation of p38 mitogen-activated protein kinase. To
determine the cardioprotective mechanism of ghrelin, we compared the roles of ghrelin and des-octanoyl ghrelin in the
present study. Des-octanoyl ghrelin is produced by removing octanoic acid on Ser3 from ghrelin, and lacks GH-releasing
activity. Both ghrelin and des-octanoyl ghrelin effectively ameliorated ISO-induced myocardial injury, cardiac function
inhibition, cardiomegaly, collagen deposition and fibrosis. The protective effect of des-octanoyl ghrelin was much weaker
than that of ghrelin, which indicates that ghrelin plays its cardioprotective role in both a GH-dependent and -independent
manner.
In summary, ISO-induced myocardial injury results in a compensatory increase in cardiac ghrelin and GHSR mRNA level
and receptor binding capacity. Administering exo-genous ghrelin and des-octanoyl ghrelin effectively attenuated
ISO-induced myocardial injury, potentiated cardiac function, and ameliorated cardiac fibrosis, but the protection afforded by
des-octanoyl ghrelin was markedly weaker than that of ghrelin, showing that ghrelin plays its cardioprotective role in a
GH-dependent and -independent manner. Ghrelin could be an endogenous cardioprotective factor and a novel target in the
prevention of and/or therapy for cardiovascular disease.
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: 656-60.
2 St-Pierre DH, Wang L, Tache Y. Ghrelin: a novel player in the gut-brain regulation of growth hormone and energy balance. News
Physiol Sci 2003; 18: 242-6.
3 Shiiya T, Nakazato M, Mizuta M, Date Y, Mondal MS, Tanaka M,
et al. Plasma ghrelin levels in lean and obese humans and the effect
of glucose on ghrelin secretion. J Clin Endocrinol Metab 2002; 87:
240-4.
4 Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P,
et al. The tissue distribution of the mRNA of ghrelin and subtypes
of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 2002; 87: 2988-91.
5 Papotti M, Ghe C, Cassoni P, Catapano F, Deghenghi R, Ghigo E,
et al. Growth hormone secretagogue binding sites in peripheral human
tissues. J Clin Endocrinol Metab 2000; 85: 3803-7.
6 Jeffery PL, Herington AC, Chopin LK. The potential autocrine/paracrine roles of ghrelin and its receptor in hormone-dependent cancer.
Cytokine Growth Factor Rev 2003; 14: 113-22.
7 Chang L, Ren Y, Liu X, Li WG, Yang J, Geng B,
et al. Protective effects of ghrelin on ischemia/reperfusion injury in the isolated rat heart.
J Cardiovasc Pharmacol 2004; 43: 165-70.
8 Chang L, Du JB, Gao LR, Pang YZ, Tang CS. Effect of ghrelin on septic shock in rats. Acta Pharmacol Sin 2003; 24: 45-9.
9 Chang L, Zhao J, Li GZ, Geng B, Pan CS, Qi YF,
et al. Ghrelin protects myocardium from isoproterenol-induced injury in rats. Acta
Pharmacol Sin 2004; 25: 113-7.
10 Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000; 407: 908-13.
11 Thompson NM, Gill DA, Davies R, Loveridge N, Houston PA, Robinson IC,
et al. Ghrelin and des-octanoyl ghrelin promote adipogenesis
directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. Endocrinology 2004; 145: 234-42.
12 Bedendi I, Alloatti G, Marcantoni A, Malan D, Catapano F, Ghe C,
et al. Cardiac effects of ghrelin and its endogenous derivatives
des-octanoyl ghrelin and des-Gln14-ghrelin. Eur J Pharmacol 2003; 476: 87-95.
13 Rona G, Chappel CI, Balzas T, Gaudry R. An infarct-like myocardial lesion and other toxic manifestations produced by isoproterenol in
the rat. Arch Pathol 1959; 67: 443-55.
14 Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95:
351-8.
15 Corongiu F, Banni S, Dessi M. Conjugated dienes detected in tissue lipid extracts by second derivative spectrophotometry. Free Radic
Biol Med 1989; 7: 183-6.
16 Young M, Fullerton M, Dilley R, Funder J. Mineralocorticoids, hypertension, and cardiac fibrosis. J Clin Invest 1994; 93: 2578-83.
17 Qi YF, Bu DF, Niu DD, Shi YR, Wang SH, Pang YZ,
et al. Effects of different peptide fragments derived from proadrenomedullin on gene
expression of adrenomedullin gene. Peptides 2002; 23: 114-7.
18 Wol AA, Hines DK, Karliner JS. Refined membrane preparations mask ischemic fall in myocardial beta receptor density. Am J Physiol
1989; 257: H1032-6.
19 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 1976; 72: 248-54.
20 Tang CS, Liu MS. Initial externalization followed by internalization of adrenergic receptors in rat heart during sepsis. Am J Physiol 1996;
270: R254-63.
21 Matsumura K, Tsuchihashi T, Fujii K, Abe I, Iida M. Central ghrelin modulates sympathetic activity in conscious rabbits. Hypertension
2002; 40: 694-9.
22 Shimizu Y, Nagaya N, Teranishi Y, Imazu M, Yamamoto H, Shokawa T,
et al. Ghrelin improves endothelial dysfunction through growth
hormone-independent mechanisms in rats. Biochem Biophys Res Commun 2003; 310: 830-5.
23 Wiley KE, Davenport AP. Comparison of vasodilators in human internal mammary artery: ghrelin is a potent physiological antagonist
of endothelin-1. Br J Pharmacol 2002; 136: 1146-52.
24 Enomoto M, Nagaya N, Uematsu M, Okumura H, Nakagawa E, Ono F,
et al. Cardiovascular and hormonal effects of subcutaneous
administration of ghrelin, a novel growth hormone-releasing peptide, in healthy humans. Clin Sci 2003; 105: 431-5.
25 Nagaya N, Miyatake K, Uematsu M, Oya H, Shimizu W, Hosoda
H, et al. Hemodynamic, renal, and hormonal effects of ghrelin infusion
in patients with chronic heart failure. J Clin Endocrinol Metab 2001; 86: 5854-9.
26 Kishimoto M, Okimura Y, Nakata H, Kudo T, Iguchi G, Takahashi Y,
et al. Cloning and characterization of the 5(V)-flanking region of
the human ghrelin gene. Biochem Biophys Res Commun 2003; 305: 186-92.
27 Li WG, Gavrila D, Liu X, Wang L, Gunnlaugsson S, Stoll LL,
et al. Ghrelin inhibits proinflammatory responses and nuclear factor-kappaB
activation in human endothelial cells. Circulation 2004; 109: 2221-6.
28 Volterrani M, Giustina A, Lorusso R, Giordano A. Does growth hormone play a role in chronic heart failure? Heart Fail Monit 2002; 3:
60-4.
29 Ryoke T, Gu Y, Mao L, Hongo M, Clark RG, Peterson KL,
et al. Progressive cardiac dysfunction and fibrosis in the cardiomyo-pathic
hamster and effects of growth hormone and angiotensin-converting enzyme inhibition. Circulation 1999; 100: 1734-43.
30 Imanishi R, Ashizawa N, Ohtsuru A, Seto S, Akiyama-Uchida Y, Kawano H,
et al. GH suppresses TGF-beta-mediated fibrosis and retains
cardiac diastolic function. Mol Cell Endocrinol 2004; 218: 137-46.
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