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Am J Physiol Heart Circ Physiol 287: H1522–H1529, 2004.
First published May 13, 2004; 10.1152/ajpheart.00193.2004.
Ghrelin induces vasoconstriction in the rat coronary
vasculature without altering cardiac peptide secretion
Chris J. Pemberton,1 Heikki Tokola,2 Zsolt Bagi,4 Akos Koller,4 Juhani Pöntinen,2 Antti Ola,2
Olli Vuolteenaho,3 István Szokodi,2,5 and Heikki Ruskoaho2
1
Christchurch Cardioendocrine Research Group, Christchurch School of Medicine, Christchurch 8001, New Zealand;
Departments of 2Pharmacology and Toxicology and 3Physiology, Biocenter Oulu, University of Oulu,
FIN-90014 Oulu, Finland; 4Department of Pathophysiology, Semmelweis University, Budapest 8015;
and 5Heart Institute, Faculty of Medicine, University of Pécs, 7624 Pécs, Hungary
Submitted 4 March 2004; accepted in final form 11 May 2004
calcium channels; protein kinases; natriuretic peptides
is a recently discovered 28-amino acid peptide containing an n-octanoyl modification at Ser3 and is the endogenous ligand for a previously identified orphan growth hormone
(GH) secretagogue receptor (GHS-R) (14). The evidence to
date suggests that ghrelin is synthesized, produced, and released from the stomach and circulates at reasonable concentrations (⬃100 pmol/l) to act on the pituitary promoting GH
release (14, 20). On the other hand, accumulating evidence
suggests that GH has actions on the myocardium to affect
GHRELIN
Address for reprint requests and other correspondence: H. Ruskoaho, Dept.
of Pharmacology and Toxicology, Faculty of Medicine, Univ. of Oulu, PO Box
5000, FIN-90014 Oulu, Finland (E-mail: [email protected]).
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growth and contractility (9) and that long-term GH therapy can
augment intracellular systolic Ca2⫹ levels in myocytes from
rats with postinfarction heart failure (34). This leads to the
possibility that agents that act through the GHS-R family of
receptors may also act on the myocardium, because these
receptors are present in the rat and human heart and vasculature
(14), and they appear to be upregulated in atherosclerosis (12).
Indeed, GHS-R populations have been detected in the heart,
and these are thought to mediate the coronary vasoconstrictor
actions of the synthetic GH-releasing hexapeptide hexarelin in
isolated heart preparations (3). Hexarelin improves cardiac
function and decreases peripheral resistance in rats with myocardial infarction (35) and also protects the isolated heart from
ventricular dysfunction associated with Ca2⫹ depletion (29).
Ghrelin may also have a role in the cardiovascular system,
because it is expressed in the heart at both the RNA (14) and
peptide (10) levels and it decreases mean arterial pressures and
increases cardiac index and stroke volume index without any
effect on heart rate in humans (21). In rats with heart failure
and cachexia, chronic ghrelin administration improves left
ventricular dysfunction and attenuates ventricular remodelling
(23). However, it is unknown whether ghrelin has direct
actions on cardiac contractility or coronary vascular tone or
whether it can alter the gene expression and/or peptide release
of cardiac-specific molecules such as atrial natriuretic peptide
(ANP) or B-type natriuretic peptide (BNP). Therefore, we
sought to determine 1) whether ghrelin possesses the ability to
modulate hemodynamics in isolated perfused heart preparations and in isolated coronary arteriole preparations, 2) whether
ghrelin could have a role in modulating cardiac endocrine
function by studying its effects on ANP and BNP secretion and
gene expression in perfused heart preparations as well as in
cultured neonatal rat ventricular myocytes, and 3) whether
ghrelin is present in cardiac tissue and, if so, is the heart a
possible source of circulating ghrelin.
METHODS
Chemicals. Synthetic rat Ser3-(n-octanoyl)ghrelin was obtained
from Phoenix Pharmaceuticals (Belmont, CA) and dissolved in perfusion buffer immediately before infusion or in DMEM/F-12 cell
culture medium supplemented with 0.1% BSA to give a 10 ␮mol/l
stock solution. Diltiazem (Orion Pharma) was initially dissolved in
0.9% saline, whereas 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6135/04 $5.00 Copyright © 2004 the American Physiological Society
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Pemberton, Chris J., Heikki Tokola, Zsolt Bagi, Akos Koller,
Juhani Pöntinen, Antti Ola, Olli Vuolteenaho, István Szokodi, and
Heikki Ruskoaho. Ghrelin induces vasoconstriction in the rat coronary vasculature without altering cardiac peptide secretion. Am J
Physiol Heart Circ Physiol 287: H1522–H1529, 2004. First published
May 13, 2004; 10.1152/ajpheart.00193.2004.—We administered ghrelin, a novel growth hormone-releasing hormone, to isolated perfused
rat hearts, coronary arterioles, and cultured neonatal cardiomyocytes
to determine its effects on coronary vascular tone, contractility, and
natriuretic peptide secretion and gene expression. We also determined
cardiac levels of ghrelin and whether the heart is a source of the
circulating peptide. Ghrelin dose dependently increased coronary
perfusion pressure (44 ⫾ 9%, P ⬍ 0.01), constricted isolated coronary
arterioles (12 ⫾ 2%, P ⬍ 0.05), and significantly enhanced the
pressure-induced myogenic tone of arterioles. These effects were
blocked by diltiazem, an L-type Ca2⫹ channel blocker, and bisindolylmaleimide (Bis), a protein kinase C (PKC) inhibitor. Interestingly, coinfusion of ghrelin with diltiazem completely restored myocardial contractile function that was decreased 30 ⫾ 3% (P ⬍ 0.01) by
diltiazem alone. In contrast, combination of ghrelin with diltiazem or
Bis did not significantly alter atrial natriuretic peptide (ANP) secretion, which was decreased 40% (P ⬍ 0.01) and 50% (P ⬍ 0.05) by
these agents alone, respectively. Administration of ghrelin to cultured
cardiomyocytes had no effect on ANP or B-type natriuretic peptide
secretion or gene expression. Detectable amounts of low-molecularweight ghrelin were present in cardiac tissue extracts but not in
isolated heart perfusate. Thus we provide the first evidence that
ghrelin has a coronary vasoconstrictor action that is dependent on
Ca2⫹ and PKC. Furthermore, the data obtained from diltiazem infusion suggest that ghrelin has a role in regulation of contractility when
L-type Ca2⫹ channels are blocked. Finally, the observation that
immunoreactive ghrelin is found in cardiac tissue suggests the presence of a local cardiac ghrelin system.
GHRELIN VASOCONSTRICTION: ROLE OF CA2⫹ AND PKC
AJP-Heart Circ Physiol • VOL
120 mmHg were measured. In the presence of 80-mmHg intraluminal
pressure, ghrelin (1 nmol/l) was then added to the vessel chamber and
circulated for 60 min while the arteriolar diameter was continuously
recorded.
In separate experiments, in the presence of 80-mmHg intraluminal
pressure, arterioles were incubated with Bis (90 nmol/l) for 30 min
(before the administration of ghrelin), and ghrelin-induced changes in
arteriolar diameter as a function of time were then obtained. Next,
changes in diameter to increases in intraluminal pressure were measured, and each pressure step was maintained for 5– 8 min to allow the
vessel to reach a steady-state diameter. Arteriolar dilations to the
Ca2⫹ antagonist diltiazem (10⫺9–10⫺5 mol/l) were also obtained
before and after incubation of arterioles with ghrelin and ghrelin plus
Bis. Ghrelin-induced arteriolar responses as a function of time are
shown as changes in arteriolar diameter. Myogenic constriction was
calculated at each pressure step as the percent change in diameter
compared with the corresponding passive diameter in Ca2⫹-free
Krebs solution. Diltiazem-induced arteriolar responses were expressed as a percentage of the maximal dilation of the vessel, defined
as the passive diameter at 80 mmHg of intraluminal pressure in
Ca2⫹-free Krebs solution. Data are expressed as means ⫾ SE.
Cell culture. Ventricular myocytes were prepared from 2- to
4-day-old neonatal rat hearts (28, 36). Cells were plated at the density
of 2 ⫻ 105 cells/cm2 onto Falcon wells 15–35 mm in diameter. After
24 h, the serum-containing medium was replaced with complete
serum-free medium (CSFM). After 48-h incubation in CSFM, the
wells were divided into test groups, and the medium was replaced
with CSFM or CSFM supplemented with 1, 10, or 100 nmol/l ghrelin
and incubated up to 48 h at 37°C. Medium was replenished every
24 h. After experiments, cells were washed twice with PBS and
quickly frozen at ⫺70°C before RNA extraction.
Isolation and analysis of RNA. Total RNA from cultured cardiac
myocytes was isolated using the guanidine thiocyanate-CsCl method
(5). For RNA Northern blot analyses, 1.8- to 6-␮g samples were
separated by electrophoresis and transferred to nylon membranes
(Osmonics). The cDNA probes complementary to rat ANP or BNP
mRNA or 18S rRNA were random prime labeled with Rediprime II
(Amersham Biosciences). The membranes were hybridized and
washed three times for 20 s at 62°C as previously described (28, 36).
Thereafter, the membranes were exposed with PhosphorImager
screens (Amersham Biosciences), which were scanned with Molecular Imager FX Pro Plus and quantitated using Quantity One software
(Bio-Rad). Hybridization signals of ANP and BNP were normalized
to that of 18S RNA.
Cardiac tissue extraction and HPLC. Cardiac tissue extracts from
ventricular stretch experiments were prepared from atrial and ventricular tissue samples as previously described (27). Extracted supernatants were subjected to a specific RIA for ghrelin to calculate tissue
concentrations. Supernatants were then dried under air, reconstituted
in 20% acetonitrile-0.1% trifluoroacetic acid (TFA), and subjected to
reverse-phase HPLC (RP-HPLC). Immunoreactive ghrelin fractions
from RP-HPLC were then pooled, concentrated, dried under air, and
reconstituted in 10% acetonitrile-0.15 mol/l NaCl for size exclusion
HPLC on a Pharmacia HR10/30 Superdex high-resolution peptide
column. Each immunoreactive fraction from RP-HPLC was run separately on size exclusion HPLC using an isocratic gradient of 10%
acetonitrile-0.1% TFA and 0.15 mol/l NaCl, with a flow rate of 0.25
ml/min. Fractions were collected at 1-min intervals and subjected to
further ghrelin RIA to establish molecular size.
Hormone RIA. Immunoreactive ANP (38) and BNP (13) concentrations in the perfusate and cell culture medium were determined
using specific RIA as previously described. The sensitivities of the
BNP and ANP assays were 2 and 1 fmol/tube, respectively. Fifty
percent displacements (ED50) of the respective standard curve occurred at 16 and 25 fmol/tube. The intra- and interassay variations
were 10% and 15%, respectively. Immunoreactive ghrelin in perfusate
and cardiac tissue extracts was measured as previously described (26).
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3-(1H-indol-3-yl)maleimide [GF-109203X, bisindolylmaleimide I
(Bis), Calbiochem] was dissolved in DMSO. The final concentration
of each solvent was ⬍0.004%. Phenylephrine (PE; Sigma) was
dissolved in DMEM/F-12 to give a 10 mmol/l stock solution. BNP
peptide and antiserum as well as a 390-bp fragment of rat BNP cDNA
probe (24) were provided by Dr. Kazuwa Nakao, Kyoto University
School of Medicine (Kyoto, Japan). The rat ANP cDNA probe was
provided by Dr Peter L. Davies, Queen’s University (Kingston,
Ontario, Canada) (6).
Isolated heart preparation. Sprague-Dawley rats (n ⫽ 64, 280 –320
g) obtained from the Center for Experimental Animals, University of
Oulu (Oulu, Finland), underwent a modified isolated heart procedure
similar to that described previously (8). The Animal Use and Care
Committee of the University of Oulu approved the experimental
design. Briefly, the animals were anesthetized with CO2, the chest was
quickly opened, and cannulation above the aortic valve allowed
perfusion at 37°C in a retrograde fashion (Langendorff) at constant
flow (13 ml/min) using an oxygenated (95% O2-5% CO2) modified
Krebs-Henseleit buffer (8). Perfusion pressure, which reflects coronary resistance, was measured by using a pressure transducer (Isotec,
Hugo Sachs Elektronik) situated on a side arm of the aortic cannula.
The left atrium was removed to allow insertion of a liquid-filled
balloon into the left ventricle, which was connected to a pressure
transducer to allow measurement of heart rate and contractile parameters. Hearts beat spontaneously and were allowed to equilibrate for
30 min. After a 10-min control period, hearts were perfused at 0.5
ml/min in a random protocol with either vehicle or ghrelin for 60 min.
To study the intracellular mechanisms underlying ghrelin-induced
vasoconstriction, we infused the L-type Ca2⫹ channel blocker diltiazem (1 ␮mol/l) and the specific PKC inhibitor Bis (90 nmol/l) either
alone or in combination with 1 nmol/l ghrelin. The concentrations of
diltiazem (32) and Bis (33) used here were calculated and titrated to
suitable doses based on previous reports indicating their effectiveness
in isolated heart preparations. Changes in heart rate, perfusion pressure, and parameters of cardiac function (left ventricular end-diastolic
pressure, developed pressure, systolic pressure, and ⫾dP/dt) were
recorded and analyzed using Ponemah data-acquisition software
(Gould Instrument Systems). A 15-ml volume of perfusate was
collected at 10-min intervals for hormone measurements by specific
radioimmunoassay (RIA).
In a separate set of experiments, we sought to determine whether
ventricular stretch could release immunoreactive ghrelin from the
myocardium. Six hearts were prepared as described above and allowed to equilibrate for 30 min. After a 10-min control period, hearts
were then stretched for 2 h by filling the intraventricular balloon to
achieve an end-diastolic pressure of ⬃25 mmHg, which we have
previously shown to stimulate BNP gene expression and release (8).
Hemodynamic variables were recorded on computerized software,
and perfusate was collected every 10 min for the analysis of ghrelin
immunoreactivity.
Responses of isolated coronary arterioles. Vascular responses to
ghrelin were investigated in isolated rat coronary arterioles, as described previously (1, 15, 16). Briefly, with the use of microsurgery
instruments and an operating microscope, a branch of the septal
coronary artery (⬃1 mm in length) was isolated and transferred into
an organ chamber containing two glass micropipettes filled with
Krebs solution equilibrated with a gas mixture of 95% O2 and 5%
CO2 at pH 7.4. Arterioles were then cannulated on both ends with
micropipettes, and inflow and outflow pressures were set to 80 mmHg
by a pressure servo-control system (Living Systems Instrumentation).
The temperature was set at 37°C by a temperature controller (Grant
Instruments). The internal arteriolar diameter at the midpoint of the
arteriolar segment was measured by videomicroscopy with a microangiometer (Texas Instruments). Changes in arteriolar diameter and
intraluminal pressure were continuously recorded and analyzed
(Biopac Systems). After 1-h incubation period, changes in arteriolar
diameter to a stepwise increase in intraluminal pressure from 20 to
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GHRELIN VASOCONSTRICTION: ROLE OF CA2⫹ AND PKC
Table 1. Basal hemodynamic variables and perfusate ANP concentrations for the 6 experimental groups before infusion
in isolated rat heart preparations
Perfusion pressure, mmHg
Ventricular pressures
Systolic, mmHg
DP, mmHg
LVEDP, mmHg
⫹dP/dt, mmHg/s
⫺dP/dt, mmHg/s
Heart rate, beats/min
ANP, pmol/l
Vehicle
Ghrelin
Dil
Bis
Ghrelin ⫹ Dil
Ghrelin ⫹ Bis
67.8⫾4.4
72.0⫾3.5
74.2⫾3.3
68.9⫾3.0
69.6⫾2.4
72.5⫾5.0
78.4⫾6.6
75.3⫾6.8
3.9⫾1.2
2,333.4⫾264.4
1,729.0⫾244.2
245.8⫾24.1
4.1⫾0.4
74.4⫾8.0
70.7⫾2.9
3.9⫾0.8
2,013.5⫾169.5
1,659.4⫾171.2
248.4⫾17.8
3.7⫾1.1
71.5⫾5.4
66.0⫾5.8
5.6⫾2.0
2,130.5⫾128.8
1,455.5⫾131.8
233.5⫾12.2
4.8⫾0.8
76.0⫾6.3
72.9⫾5.5
3.8⫾0.5
2,335.5⫾424.7
1,743.9⫾285.0
264.3⫾10.8
5.1⫾0.6
68.3⫾6.2
65.6⫾6.1
3.2⫾0.9
1,846.2⫾156.5
1,325.7⫾156.6
244.3⫾17.2
4.8⫾0.7
67.4⫾7.8
64.6⫾7.5
3.1⫾0.8
2,129.3⫾273.8
1,478.1⫾182.2
245.0⫾10.3
4.4⫾0.4
Values are means ⫾ SE; n ⫽ 6 heart preparations/group. The dose of ghrelin was 1 nmol/l. Dil, diltiazem (1 ␮mol/l); Bis, bisindolylmaleimide (90 nmol/l);
DP, developed pressure; LVEDP, left ventricular end-diastolic pressure; ⫾dP/dt, developed pressure over time; ANP, atrial natriuretic peptide.
RESULTS
Infusion of ghrelin in isolated perfused rat hearts. Basal
values for hemodynamic variables in isolated hearts are given
in Table 1. One-hour infusion of ghrelin had no significant
effect on cardiac contractility at the doses of 0.01–10 nmol/l,
although there was a tendency for developed pressure to slowly
decrease in the ghrelin group compared with the vehicle group.
Perfusion pressure, however, increased significantly in a dosedependent manner during ghrelin infusion. The effect was most
pronounced at the concentration of 1 nmol/l, which resulted in
a 44 ⫾ 9% increase in perfusion pressure compared with the
vehicle group (P ⬍ 0.01; Fig. 1).
Role of L-type Ca2⫹ channels and PKC. To study potential
signaling mechanisms underlying ghrelin-induced increases in
Fig. 1. Dose-dependent vasoconstrictive effect of ghrelin (Ghr) in isolated
perfused rat hearts. The results are expressed as the percent change in perfusion
pressure (PP) relative to basal values. Ghrelin infusion at 0.1 and 1 nmol/l
increased coronary PP compared with vehicle infusion. †P ⬍ 0.01, ghrelin
versus vehicle; n ⫽ 6 [two-way ANOVA for repeated measures, followed by
a least-significant difference (LSD) post hoc test].
AJP-Heart Circ Physiol • VOL
coronary perfusion pressure, we infused the L-type Ca2⫹
channel blocker diltiazem and the specific PKC inhibitor Bis
with 1 nmol/l ghrelin. Ghrelin-induced increases in perfusion
pressure were completely abolished by a coinfusion of 1
␮mol/l diltiazem (P ⬍ 0.01; Fig. 2A) and 90 nmol/l Bis (P ⬍
0.05; Fig. 2B). Diltiazem (Fig. 2A) or Bis (Fig. 2B) had no
significant effect on perfusion pressure when infused alone.
As shown in Fig. 2C, 1 nmol/l ghrelin had no significant
effect on developed pressure when administered alone. However, the negative inotropic effect of diltiazem (⫺30 ⫾ 3%,
P ⬍ 0.01 vs. vehicle) was completely abolished when ghrelin
was coinfused (P ⬍ 0.05 vs. diltiazem), restoring developed
pressure to vehicle control levels (Fig. 2C). Bis had no effect
on developed pressure either when infused alone or in combination with ghrelin (data not shown).
Responses of isolated coronary arterioles. After a 1-h incubation period, the active diameter of coronary arterioles was
103 ⫾ 9 ␮m in the presence of 80-mmHg pressure. The
administration of 1 nmol/l ghrelin resulted in a slow constriction of coronary arterioles (P ⬍ 0.05; Fig. 3A). Furthermore,
ghrelin significantly enhanced the pressure-induced myogenic
tone of arterioles between 40 and 120 mmHg (P ⬍ 0.05; Fig.
3B). Both effects were attenuated with 90 nmol/l Bis (Fig. 3, A
and B). Finally, diltiazem-induced arteriole vasodilation was
not altered by ghrelin nor ghrelin and Bis together (Fig. 3C).
Effect of ghrelin on natriuretic peptide secretion in isolated
perfused hearts. Basal ANP concentrations in the isolated heart
perfusate before agent infusions are given in Table 1. Ghrelin
infusion at 1 nmol/l had no effect on basal ANP secretion
compared with vehicle (P ⫽ 0.24) in the isolated heart preparation. Neither did ghrelin modify ANP secretion when coinfused with diltiazem. Diltiazem alone caused a 40 – 45% reduction (P ⬍ 0.01) in ANP secretion (Fig. 4A), an observation
consistent with previous reports (27). Bis caused a significant
50% (P ⬍ 0.05) decrease in perfusate ANP levels (Fig. 4B),
which was not significantly affected by coinfusion with ghrelin
(P ⫽ 0.37, Bis vs. Bis ⫹ ghrelin). Yet, the previous statistically
significant difference between Bis and vehicle disappeared
when ghrelin was coinfused with Bis (P ⫽ 0.07, Bis ⫹ ghrelin
vs. vehicle). BNP secretion was unaffected in all isolated heart
experiments (data not shown).
Ghrelin and natriuretic peptide secretion and gene expression in cultured cardiomyocytes. Cultured neonatal rat ventricular myocytes were treated with incremental doses (1–100
nmol/l) of ghrelin for up to 48 h. PE, an ␣-adrenergic agonist
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The RIA has a mean zero binding of 24 ⫾ 2%, mean sample detection
limit of 3.3 fmol/tube, and ED50 of 136.2 ⫾ 10 fmol/ml.
Statistical analysis. Results are presented as means ⫾ SE. Hemodynamic and peptide RIA time-course data from isolated heart and
coronary arteriole experiments were analyzed with two-way ANOVA
for repeated measures, followed by a least-significant difference
(LSD) post hoc test. For multiple comparisons, data were analyzed
with one-way ANOVA, followed by a LSD post hoc test. Gene
expression data were analyzed using Student’s t-test for unpaired data.
A value of P ⬍ 0.05 was considered statistically significant.
GHRELIN VASOCONSTRICTION: ROLE OF CA2⫹ AND PKC
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However, these levels were 450- to 1,800-fold less than those
reported for stomach tissue extracts (10, 14). Primary analysis
of ghrelin immunoreactivity on RP-HPLC revealed two peaks
in both atrial and ventricular extracts (Fig. 7). Peak 1 (Fig. 7,
A and C) was consistent in RP-HPLC retention time with
synthetic octanoyl ghrelin, whereas peak 2 eluted later. Separate analysis of both atrial and ventricular peaks by size
exclusion HPLC revealed each single peak to be of low
molecular weight (Mr ⬃3,400; Fig. 7, B and D). Despite
cardiac tissue extracts containing bona fide ghrelin, immunoreactive ghrelin was not detectable at any time in the perfusate
from stretch experiments, arguing against stretch-mediated
cardiac secretion (data not shown).
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Fig. 2. A and B: coinfusion of 1 ␮mol/l diltiazem (A) or 90 nmol/l bisindolylmaleimide (Bis; B) with 1 nmol/l ghrelin completely abolished the vasoconstrictive effect reflected by increased PP in isolated perfused rat hearts,
whereas diltiazem and Bis alone had no effect on coronary vascular resistance.
*P ⬍ 0.05, ghrelin vs. vehicle; ††P ⬍ 0.01, ghrelin ⫹ diltiazem vs. ghrelin;
†P ⬍ 0.05, ghrelin ⫹ Bis vs. ghrelin. C: the negative inotropic effect of
diltiazem (**P ⬍ 0.01 vs. vehicle) was totally blocked by coinfusion of ghrelin
(#P ⬍ 0.05 vs. diltiazem alone), which alone had no effect on developed
pressure (DP) (two-way ANOVA for repeated measures followed by a LSD
post hoc test).
known to stimulate ANP and BNP synthesis and secretion in
cardiac myocytes (28, 36), was used as a positive control.
Administration of 10 ␮mol/l PE stimulated ANP and BNP
secretion by 77 ⫾ 18% (P ⬍ 0.001; Fig. 5A) and 93 ⫾ 11%
(P ⬍ 0.05; Fig. 5B), respectively, and resulted in 70% (P ⬍
0.05) increases in both ANP (Fig. 6B) and BNP (Fig. 6C)
mRNA levels. Ghrelin had no significant effect on ANP or
BNP peptide secretion (Fig. 5) and gene expression (Fig. 6).
Cardiac tissue ghrelin immunoreactivity and response to
ventricular stretch. Cardiac tissue extracts contained immunoreactive ghrelin, with the right atrium having a slightly higher
content compared with the right and left ventricle (4.1 ⫾ 0.5,
2.4 ⫾ 0.1, and 1.0 ⫾ 0.1 fmol/mg wet wt, respectively, n ⫽ 6).
AJP-Heart Circ Physiol • VOL
Fig. 3. A: ghrelin-induced constriction of isolated coronary arterioles was
attenuated by 90 nmol/l Bis (*P ⬍ 0.05, ghrelin vs. control; ⫹P ⬍ 0.05,
ghrelin ⫹ Bis vs. ghrelin; both n ⫽ 5). B: ghrelin enhancement of myogenic
constriction of coronary arterioles was also attenuated by 90 nmol/l Bis (*P ⬍
0.05 and ⫹P ⬍ 0.05 as in A; n ⫽ 5). C: diltiazem-induced arteriolar dilations
were not altered by ghrelin and ghrelin ⫹ Bis (n ⫽ 5) (two-way ANOVA
followed by Tukey’s post hoc test).
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GHRELIN VASOCONSTRICTION: ROLE OF CA2⫹ AND PKC
Fig. 4. A: the 1 ␮mol/l diltiazem-induced decreases in perfusate atrial natriuretic peptide concentration ([ANP]) were not altered by coinfusion of 1
nmol/l ghrelin in isolated perfused rat hearts. B: the significant decrease in
perfusate [ANP] seen during 90 nmol/l Bis infusion was not observed during
coinfusion with ghrelin. *P ⬍ 0.05 vs. vehicle; †P ⬍ 0.01 vs. vehicle (two-way
ANOVA for repeated measures followed by a LSD post hoc test).
DISCUSSION
Ghrelin was initially discovered from the stomach and identified as the natural ligand for a particular G protein-coupled
orphan receptor, denoted GHR-S (14). Subsequent studies have
clearly shown ghrelin to be a potent stimulator of GH release
(21, 40) and that it has a significant role in energy balance and
carbohydrate metabolism (37). However, more recent work has
shown ghrelin to have effects on blood pressure, cardiac
function, and energetics (23), particularly with respect to cardiac catabolic-anabolic imbalance in severe congestive heart
failure (22). These results are supported by the identified tissue
distributions of GHR-S, which include the lung, intestine,
pancreas, and adipose tissue (14) and also the heart and
coronary vasculature (12). However, although ghrelin has been
implicated as a potential cardiovascular peptide, it is unknown
whether it can directly influence cardiac function or coronary
vasomotor tone or whether it has any effect on the endocrine
function of the heart, such as natriuretic peptide secretion. Thus
this report provides several notable firsts: 1) we describe a
constrictor effect of ghrelin on the coronary vasculature and its
dependence on Ca2⫹ and PKC; 2) there is evidence of a role for
ghrelin in modulating cardiac contractile function in relation to
Ca2⫹ status; 3) there is a the lack of effect of ghrelin on ANP
AJP-Heart Circ Physiol • VOL
Fig. 5. Effect of ghrelin on ANP (A) and B-type natriuretic peptide (BNP; B)
secretion in cultured neonatal rat ventricular cells. Open bars, control; light
shaded bars, 1 nmol/l ghrelin; medium shaded bars, 10 nmol/l ghrelin; dark
shaded bars, 100 nmol/l ghrelin; hatched bars, 10 ␮mol/l phenylephrine (PE).
Results are expressed as the percent change in immunoreactive (ir)-BNP or
ir-ANP secretion (n ⫽ 14 –54, 7 independent cultures). Basal 24-h accumulation of ANP and BNP into the culture medium in the control cells was 18.4 ⫾
3.7 and 3.4 ⫾ 0.5 pmol/ml, respectively. *P ⬍ 0.05 and ***P ⬍ 0.001 vs.
control at each time point (one-way ANOVA followed by a LSD post hoc test).
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and BNP activity in cardiac myocytes; and 4) this is the first
description of whether the heart could secrete ghrelin.
The time course of the increases in perfusion pressure and
coronary arteriole constriction in response to ghrelin observed
here was slow. This effect was significantly inhibited by both
diltiazem and Bis, suggesting a dependence on both Ca2⫹ and
PKC, respectively. In this regard, ghrelin has an almost identical profile to previously described hexarelin-induced increases in coronary perfusion pressure (3) but is effective at
1,000-fold lower doses. Such a high potency for activity
(observed at ⬃1 nmol/l in the present study) is comparable
with endothelin-1 (ET-1) (31), suggesting that ghrelin is one of
the most potent identified regulators of coronary tone.
Initial dose-ranging experiments have revealed that doses
lower and higher than 1 nmol/l ghrelin resulted in (nonsignificant) peak increases in perfusion pressure by 30 min. In
contrast, data from human studies suggest that ghrelin has in
vivo vasorelaxant activity (20, 25) that may be independent of
NO activity (25) and that it is able to antagonize ET-1-induced
vasconstriction (39) in vitro in endothelium-denuded internal
mammary artery preparations. Naturally, in vivo release of GH
from the anterior pituitary (but absent in the isolated perfused
heart), differential GHS-R distributions across tissue beds (14),
GHRELIN VASOCONSTRICTION: ROLE OF CA2⫹ AND PKC
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Fig. 6. A: Northern blot analysis of 24-h ghrelin or PE treatment on ANP or
BNP gene expression in cultured neonatal rat ventricular cells. B and C: bar
graphs showing the effect of ghrelin at the doses of 1 nmol/l (light shaded
bars), 10 nmol/l (medium shaded bars), and 100 nmol/l (dark shaded bars) on
ANP (B) and BNP (C) mRNA levels. Open bars, control; hatched bars, 10
␮mol/l PE. Results are expressed as the ratio of specific mRNA to 18S RNA
(n ⫽ 4 –7, 4 independent cultures). *P ⬍ 0.05 vs. control (Student’s t-test).
and regulatory and counterregulatory systems may account for
these observed differences. Thus Wiley and Davenport (39)
employed ghrelin in vitro at doses up to 300 nM, nearly 300
times those employed in our study, whereas Okumura et al.
(25) utilized sequential pharmacological boli (2, 5, and 10 ␮g)
of ghrelin and only achieved significant in vivo forearm vasodilation at 5- and 10-␮g doses. Given that our vasconstriction
was achieved at physiological levels of ghrelin (between 0.3
Fig. 7. Reverse-phase (A and C) and size exclusion (B and D) HPLC analysis
of immunoreactive ghrelin in atrial and ventricular tissue extracts. Two peaks
of immunoreactive ghrelin were detected by reverse-phase HPLC in both atrial
(A) and ventricular (C) extracts. Peak 1 eluted consistent with synthetic
octanoyl ghrelin, whereas peak 2 eluted later. Size-exclusion HPLC revealed
identical low-molecular-weight forms in atrial (B) and ventricular (D) extracts
(calculated Mr ⬃3,400). Synthetic ghrelin and molecular-weight standards are
indicated by arrows. IR ghrelin, immunoreactive ghrelin per fraction (pmol/l).
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GHRELIN VASOCONSTRICTION: ROLE OF CA2⫹ AND PKC
AJP-Heart Circ Physiol • VOL
mechanism. Indeed, ghrelin-induced vasoconstriction was
completely abolished by either L-type Ca2⫹ channel blockade
or PKC inhibition. Thus the vasoconstrictive and endocrine
effects of ghrelin might be mediated through different GHS-R
subtypes (3) as well as different isozymes of PKC (7). In
support of this are the observations that different PKC
isozymes (some of which are Ca2⫹-dependent) are differentially distributed within cardiovascular tissues (18) and that
specific PKC isozyme anchoring protein receptors for activated
C kinase are differentially activated within cardiac myocytes
(19).
Cardiac tissue extracts contained measurable amounts of
ghrelin, with the atrium containing approximately twice as
much as the ventricle on a picomole per wet weight basis,
values that are in agreement with those previously reported in
rat cardiac tissue (10). RP-HPLC and size exclusion HPLC
analysis demonstrated this immunoreactivity to be made up of
two low-molecular-weight forms of ghrelin, one of which was
consistent with the octanoyl form. This may represent multiple
posttranslational products of ghrelin processing (defined by the
degree of acylation) as recently described in human plasma and
the stomach (11), and it is known that the acyl and des-acyl
forms of the peptide have markedly different retention times on
RP-HPLC (14). However, despite the presence of quantifiable
amounts of ghrelin in cardiac tissue extracts, we could not
detect immunoreactive ghrelin in isolated heart perfusates,
despite a 10-fold concentration when prepared for RIA. This
suggests that if ghrelin has an endogenous role in modulating
cardiac function, it might act in a paracrine/autocrine manner
similar to angiotensin II and ET-1 (31).
In summary, we provide the first report of a slow-acting,
vasoconstrictor action of the novel peptide ghrelin on the
coronary vasculature that is dependent on L-type Ca2⫹ channel
and PKC activation. Additionally, the negative inotropic action
of diltiazem was effectively blocked by ghrelin, which suggests that ghrelin has a role in the regulation of cardiac Ca2⫹
homeostasis. Decreases in ANP secretion induced by blocking
L-type Ca2⫹ channels or PKC were not affected by coadministration of ghrelin, suggesting a differential action on hemodynamics versus the endocrine function of the heart. Cardiac
myocyte culture demonstrated a lack of effect of ghrelin on
natriuretic peptide secretion and gene expression, even after
48 h of administration. We show that bona fide ghrelin exists
in cardiac tissue, yet levels are not high enough to suggest that
the heart is a significant source of circulating ghrelin, which
was confirmed by the observation that ghrelin was undectable
in isolated heart perfusate and that cardiac stretch was not
sufficient to induce its release. However, when coupled with
previous reports identifying 1) the presence of GHS-Rs in
cardiac endothelial cells (12, 14) and 2) beneficial effects of
ghrelin administered to patients with heart failure (21), our
observations suggest that the cardiac ghrelin system is a potential target for future therapeutic strategies in congestive
heart failure. The precise cellular distribution of a cardiac
ghrelin system and the identification of intracellular signaling
pathways underlying these diverging actions are therefore
logical targets for further studies.
ACKNOWLEDGMENTS
We thank Marja Arbelius and Tuulikki Kärnä for expert technical assistance.
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and 1 nM), this may explain some of the discrepancy. Furthermore, in human studies by Nagaya et al. (20), the decrease in
mean arterial pressure was noted in response to a single bolus
of ghrelin, which achieved a pharmacological plasma level of
⬃45 nmol/l. Taken together, these results suggest that any
vasoconstrictor activity attributable to ghrelin may depend on
the site of administration, the dosage employed, GHS-R expression profiles, and the species in question.
Our data suggesting that ghrelin has no direct inotropic
effect on the heart is consistent with a previous report (23)
indicating no effect of the peptide on fractional cell shortening
in isolated myocytes. An intriguing aspect of our data is the
observation that ghrelin appears to protect cardiac function
when cytosolic Ca2⫹ concentration is decreased and that PKC
appears to play no significant role. Thus, when the isolated
perfused heart preparation was subjected to L-type Ca2⫹ channel blockade, the developed pressure significantly decreased
(as expected), yet coinfusion of ghrelin with diltiazem restored
developed pressure to vehicle control levels. In this context, in
vivo subcutaneous administration of hexarelin for 7 days has
been reported to precondition and protect subsequent isolated
perfused heart preparations against calcium overload-induced
increases in left ventricular end-diastolic pressure in normal
rats subjected to low Ca2⫹ perfusion/normal Ca2⫹ reperfusion
(29) or to ischemia-reperfusion injury in hypophysectomized
rats (17). Furthermore, in vivo evidence of hexarelin-induced
improvements in cardiac function have been reported after
bolus injection in humans (2), and in vitro data from isolated
rat heart preparations suggest that ghrelin may be protective
against ischemia-reperfusion injury, at least partially through
reducing myocyte lactate dehydrogenase and myoglobin release (4) and/or via PKC-related mechanisms (7). The mechanism(s) behind the protective effects of hexarelin/ghrelin is
unclear, but any improvements in cardiac function need to be
weighed up against potential deleterious constrictor actions at
higher doses of ghrelin (⬎0.7 nM) and hexarelin (⬎0.5 ␮M).
Nevertheless, the possibility that ghrelin and hexarelin may
have direct effects on Ca2⫹ homeostasis and whether this is
responsible for the beneficial effects of each peptide in experimental myocardial infarction (35) and congestive heart failure
(23) merit further investigation.
In our hands, ghrelin exhibited no effect on basal ANP or
BNP peptide secretion from isolated hearts, and it had no effect
on diltiazem-induced reductions in ANP secretion. Consistent
with the known role of PKC in regulating ANP secretion (30),
infusion of the PKC inhibitor Bis (which attenuates ␣-, ␤-, ␥-,
␦-, and ⑀-isoforms of the enzyme) significantly inhibited ANP
secretion in the isolated perfused heart preparation. Although
the difference between Bis versus Bis and ghrelin was not
significant, the effect of Bis appeared to be attenuated toward
the end of the perfusion period by coinfusion of ghrelin. This
suggests that mechanisms governing the effects of ghrelin on
cardiac hemodynamics and any putative endocrine secretory
effects of ghrelin are dissociated and/or have little influence on
one another, at least in the 1-h time period of infusion used
here. In this regard, the peptide secretion and gene expression
results from cultured ventricular cardiomyocytes indicate more
clearly the lack of effect of ghrelin on natriuretic peptide
secretion during a longer period of administration (up to 48 h).
Our results do not rule out that ghrelin may affect both
pathways (Ca2⫹ and PKC) through a common intracellular
GHRELIN VASOCONSTRICTION: ROLE OF CA2⫹ AND PKC
GRANTS
This study was supported by the Foundation of Research, Science and
Technology of New Zealand, Academy of Finland, Hungarian Scientific
Research Fund Grants T033117 and F035213, the Sigrid Juselius Foundation,
and the Finnish Foundation for Cardiovascular Research. C. J. Pemburton was
the recipient of a Postdoctoral Fellowship from the Foundation of Research,
Science and Technology of New Zealand.
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