© 1999 American Heart Association, Inc.
Molecular Medicine |
Identification and Characterization of a New Growth Hormone–Releasing Peptide Receptor in the Heart
From the Faculty of Pharmacy (V.B., J.F.B., N.M., D.L., H.O.), Université de Montréal, Montreal, Canada; Department of Pharmacology (E.E.), Université de Sherbrooke, Sherbrooke, Canada; Faculty of Veterinary Medicine (P.C.), Université de Montréal, Saint-Hyacinthe, Canada; Department of Internal Medicine (E.G.), University of Turin, Italy; Pharmacia and Upjohn (T.S.), Stockholm, Sweden; and Montreal Heart Institute (M.G.S.), Montreal, Canada.
Correspondence to Dr Huy Ong, Faculty of Pharmacy, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montreal, Quebec, H3C 3J7, Canada. E-mail ongh{at}ere.umontreal.ca
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Abstract |
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Abstract—Hexarelin, a synthetic hexapeptide of the growth hormone–releasing peptide (GHRP) family with strong growth hormone (GH)–releasing activity, features protecting activity against postischemic ventricular dysfunction in hearts from GH-deficient and senescent rats. To document whether hexarelin action is mediated through specific cardiac receptors, perfusion of Langendorff rat hearts with hexarelin and binding studies were carried out. In the Langendorff rat heart system, hexarelin induced a dose-dependent increase in coronary perfusion pressure. Nifedipine, chelerythrine, and bisindolylmaleimide partially inhibited the vasoconstriction induced by hexarelin, suggesting that this effect was mediated at least in part by L-type Ca2+ channels and protein kinase C. In contrast, diclofenac and 1-(7-carboxyheptyl)imidazole were without effect, suggesting that prostaglandins and thromboxanes were not involved in the coronary vasoconstriction induced by hexarelin. To characterize the hexarelin binding sites in the rat heart, [125I]Tyr-Bpa-Ala-hexarelin was used as photoactivatable radioligand in saturation and competitive binding studies. We specifically labeled a hexarelin receptor with an Mr of 84 000 in rat cardiac membranes. Saturation binding curves revealed a single class of binding sites with a Kd of 14.5 nmol/L and a density of 91 fmol/mg of protein. Competition binding studies gave an IC50 of 2.9 µmol/L for hexarelin; MK-0677 and EP51389, both potent GH secretagogues, did not displace the binding of the photoactivatable derivative from rat cardiac membranes. Interestingly, both compounds were devoid of any vasoconstrictive activity. These results suggest the existence of a new class of hexarelin receptor in the heart, whose role in the regulation of the coronary vascular tone is yet to be determined.
Key Words: growth hormone–releasing peptide • receptor • heart • photoaffinity labeling • Langendorff perfused heart
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Growth hormone–releasing peptides (GHRPs), which consist of a family of small synthetic peptides modeled from Met-enkephalin,1 and their nonpeptidyl derivatives2 have been shown to release growth hormone (GH) both in vitro and in vivo by direct action on the pituitary gland.3 4 The GH releasing action of GHRPs is mediated through the interaction of these ligands with a G protein–coupled receptor, which has been cloned recently from porcine, human, and rat libraries,5 6 7 as well as with their receptor subtype identified by the photolabeling approach.8 Besides this stimulatory effect on GH secretion, it has been demonstrated that hexarelin, an hexapeptide member of the GHRP family, features protective activity against postischemic dysfunction of perfused hearts isolated from GH-deficient9 and senescent rats10 receiving the peptide subcutaneously as a 2-week pretreatment. This beneficial effect induced by hexarelin is not followed by any apparent stimulation of the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis. The effect is not likely related to the cardiovascular actions of GH/IGF-1 through interaction with specific receptors expressed at the myocardial level,11 12 suggesting a direct influence of hexarelin on cardiac function. The primary aims of the present study were to evaluate whether hexarelin exerts a direct action on the cardiac function using the perfused Langendorff heart and to document the cellular signaling pathways involved. The secondary aim was to identify and to characterize the new GHRP receptor from the rat heart by the photoaffinity labeling approach using a photoactivatable derivative of hexarelin.
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Materials and Methods |
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Experimental Protocol With Langendorff Hearts
Male Sprague-Dawley rats (300 to 325 g) were purchased from Charles River (Montreal, Canada). Animal use was in accordance with the Canadian council on animal care guidelines. Rats were necrotized with CO2 until a complete loss of consciousness and promptly decapitated. Their heats were quickly excised and mounted on the experimental Langendorff setup as described by Bouchard et al.13 A dose-response curve to hexarelin (10 nmol/L to 30 µmol/L) was charted by successive infusions of increasing concentrations of the peptide. The infusions were maintained for 5 to 10 minutes, which was enough to reach steady state. A washout period of 10 minutes was allowed after the first dose-response curve. A second dose-response curve for the same agent was drawn 30 minutes after beginning an infusion with either diclofenac (1 µmol/L), 1-(7-carboxyheptyl) imidazole (1-7 CHI, 10 µmol/L), chelerythrine (1 µmol/L), bisindolylmaleimide I, HCl (1 µmol/L), nifedipine (1 µmol/L), or their respective vehicles. Drug-induced changes in coronary resistance were evaluated by computing the percentage of change in coronary perfusion pressure, measured immediately before each drug infusion and after a new steady state.
Membrane Preparations
All animals were anesthetized with sodium
pentobarbital, and their hearts were promptly removed and placed in
ice-cold saline buffer. Cardiac membranes were prepared according to
Harigaya et al.14 Membranes from bovine anterior
pituitaries were prepared according to Ong et al.8
Receptor Binding and Photolabeling With
[125I]-Tyr-Bpa-Ala-Hexarelin
The radioiodination procedure of the photoactivatable ligand and
the receptor binding assays were performed as described by Ong et
al.8 For saturation binding assays, incubations were set
in the presence of increasing concentrations of the radiolabeled
photoactivatable hexarelin derivative (from 1.5 to 45 nmol/L).
Nonspecific binding was defined as binding not displaced by 10
µmol/L hexarelin. For competition binding assays, incubations were
performed in the presence of a fixed concentration of
radioiodinated photoactivatable hexarelin derivative (0.33
nmol/L) and increasing concentrations of competitive ligands from 1
nmol/L to 10 µmol/L. After autoradiography,
bands corresponding to the specifically labeled protein of
Mr 84 000 were cut out, and radioactivity
was counted in a 
counter to establish saturation and
competition curves.
Deglycosylation of Photolabeled GHRP Receptor With
N-Glycosidase F
Deglycosylation of the photolabeled GHRP receptors from cardiac
and pituitary membranes was performed as previously
described.8
Autoradiographic Distribution of GHRP Binding Sites in
the Rat Heart
Rat hearts were processed for cryostat sectioning. Consecutive
6-µm-width sections were mounted on gelatinized glass slides.
Sections were stained with hematoxylin-eosin for
histological determination. Consecutive unstained
sections were rehydrated by incubation with PBS. Sections used for the
total binding were incubated with
[125I]Tyr-Bpa-Ala-hexarelin (0.3 nmol/L) for 60
minutes at room temperature. The nonspecific binding was determined in
sections incubated in the presence of 10 µmol/L hexarelin. All
the sections were then exposed to UV light for 15 minutes and washed
with PBS. Tissue sections were placed in cassettes for
autoradiography. The densitometry analysis of
the autoradiogram was performed using Quantity One
software (PDI) for the quantification of the total and nonspecific
binding.
Data Analysis
The effects of the different drugs on hexarelin-induced
vasoconstriction and the data analysis of densitometry were
performed by ANOVA. Radioligand saturation and competition
curves were analyzed with ALLFIT for Windows
software.15 16
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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Change in Coronary Resistance Induced by GHRPs
Figure 1
summarizes the
dose-response curves for changes in the coronary resistance at
various concentrations of peptidyl and nonpeptidyl GHRP derivatives
infused. Coronary resistance measured just before hexarelin
infusion was 5.1±0.4 mm Hg ·
min-1 · mL-1 for a
coronary flow rate of 6.8±0.7 mL ·
min-1 · g-1. At a
concentration of 30 µmol/L, hexarelin infusion induced an
increase in coronary resistance at 11.0±0.7 mm Hg
· min-1 · mL-1
for a coronary flow rate of 6.8±0.7 mL ·
min-1 · g-1.
Coronary perfusion pressure (+120%) was increased maximally at
30 µmol/L hexarelin. The increase in the coronary
resistance induced by hexarelin contrasted with that obtained with
EP51389, a truncated GHRP derivative, and the peptidomimetic MK-0677,
as maximal coronary resistance was obtained at 8.6±0.9 and
6.7±0.4 mm Hg · min-1 ·
mL-1 for EP51389 and MK-0677, respectively.
Hexarelin did not affect left ventricular
end-diastolic pressure. However, both
dP/dtmax and dP/dtmin were
reduced by 
40% at the highest concentration of hexarelin studied
(data not shown). Heart rate was not modified even by the highest dose
of hexarelin (data not shown).
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, n=3), and EP51389 (
, n=3), and perfusion pressure was
monitored to calculate coronary perfusion resistance.
Signaling Pathways Involved in the Change of Coronary
Resistance
The role of Ca2+ influx in the increase of
coronary resistance induced by hexarelin was assessed after
preperfusion with 1 µmol/L nifedipine, an L-type
Ca2+ channel inhibitor.
Nifedipine evoked a significant decrease in
coronary resistance increased by 30 µmol/L hexarelin.
The changes in the coronary perfusion pressure were (
%)
36.1±5.4 and 127.2±7.8 in the presence and absence of 1 µmol/L
nifedipine, respectively, as shown in Figure 2.
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The role of protein kinase C (PKC) in mediating the increase in
coronary resistance was evidenced by a selective
inhibitor of PKC, chelerythrine.17 This
compound significantly reduced the coronary perfusion pressure
induced by 30 µmol/L hexarelin. The changes in coronary
perfusion pressure were (%) 76.5±2.4 and 124.4±3.0 in the
presence and the absence of 1 µmol/L chelerythrine,
respectively, as shown in Figure 2
. The role of PKC was
confirmed by the use of the highly selective inhibitor of
this enzyme, bisindolylmaleimide I, HCl.18 At a
concentration of 1 µmol/L, this compound significantly reduced
the coronary vasoconstriction induced by 10 µmol/L
hexarelin (changes in coronary perfusion pressure were (%)
17.6±7.6 and 123.0±14.0 in the presence and the absence of the
inhibitor respectively, n=4).
To rule out the contribution of contractile prostaglandins
and thromboxanes in the coronary vasoconstriction
induced by hexarelin, isolated rat hearts were preperfused with 1
µmol/L diclofenac, a cyclooxygenase
inhibitor, or 10 µmol/L 1-(7-carboxyheptyl)
imidazole (1-7 CHI), an inhibitor of
thromboxane synthase.19 Neither compound
significantly affected the coronary vasoconstriction induced by
hexarelin (Figure 2).
Covalent Photolabeling of GHRP Binding Sites With
[125I]-Tyr-Bpa-Ala-Hexarelin
Covalent photolabeling of rat cardiac membranes with
[125I]-Tyr-Bpa-Ala-hexarelin revealed a single
specific binding site with an Mr of 84 000
visualized by autoradiography after SDS-PAGE (Figure 3, top). The saturation isotherm
generated by the radioactive signal detected in the band at
Mr 84 000 is reported in Figure 3, bottom. It is best fitted to a single class of binding sites with a
dissociation constant of 14.5 nmol/L and an estimate for the number of
binding sites of 91 fmol/mg of protein.
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The specificity of covalent photolabeling of rat cardiac membranes with
the photoactivatable hexarelin derivative was assessed by competitive
binding studies with hexarelin, its truncated analog EP51389, and the
nonpeptidyl derivative MK-0677, as shown in Figure 4, top. Analysis of the
competition curves gave IC50 values of 2.9
µmol/L for hexarelin and more than 10 µmol/L for EP51389 and
MK-0677. These results contrasted with those obtained with the same
competitive ligands for binding of the photoactivatable
radioligand to the GHRP receptor subtype in bovine anterior
pituitary membranes, which featured an Mr
of 57 000.8 The IC50 of
hexarelin, EP51389, and MK-0677 inhibiting the covalent binding of
[125I]-Tyr-Bpa-Ala-hexarelin to the pituitary
GHRP receptor was 0.6, 1.5, and 20 µmol/L, respectively (Figure 4, bottom). All three ligands, which are potent growth hormone
secretagogues, appeared to bind to GHRP binding sites in rat cardiac
membranes with a lower affinity than that observed for pituitary GHRP
binding sites. Interestingly, the tripeptide analog EP51389, which
displaced the radioligand binding with the same
IC50 as hexarelin in bovine pituitary membranes,
was without effect in rat cardiac membranes.
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It is well-known that a network of nonadrenergic,
noncholinergic perivascular nerve fibers supplying the coronary
vasculature releases vasoactive neuropeptides, as demonstrated in
immunocytochemical studies.20 The main neuropeptides
identified in nerves associated with coronary blood vessels,
namely neurotensin, calcitonin gene-related peptide (CGRP), substance
P, and neuropeptide Y (NPY), did not inhibit the covalent photolabeling
of the Mr 84 000 protein by the
photoactivatable derivative of hexarelin, as shown in Figure 5. Furthermore, the vasoactive peptidic
hormones endothelin 1, angiotensin II, and
arginine-vasopressin, for which specific receptors are found within the
heart, did not compete with the photolabeling of hexarelin binding
sites (angiotensin II and arginine-vasopressin; data not
shown).21 22 23
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Deglycosylation of GHRP Binding Sites
Figure 6 illustrates that incubation
of the photolabeled receptor from cardiac membrane preparations with
N-glycosidase F for 24 hours at 25°C increased its
mobility from Mr 84 000 to 57 000 (Figure 6B). The photolabeled receptor from bovine pituitary membranes
submitted to the same incubation conditions displayed only a slight
increase in mobility from Mr 57 000 to 53 000
(Figure 6A). The ability of N-glycosidase F to reduce
the apparent molecular size of the photolabeled receptor from both
cardiac and pituitary membrane preparations confirmed the
glycoprotein nature of GHRP receptors.
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Distribution of GHRP Receptors in the Heart and Among
Species
The compartmental distribution of GHRP receptor in the rat heart
has been documented. As shown in Figure 7, the density of the signal
corresponding to the specifically photolabeled protein was found to be
higher in ventricles than in atria. Cardiac GHRP receptors were not
only expressed in cardiac membranes from the rat but also in the hearts
of hamsters, dogs, and pigs as revealed in Figure 8. Although the unique
photoaffinity-labeled band at Mr 84 000 was
detected in membranes from rat and hamster hearts, membrane
preparations from canine and porcine hearts featured two specific
photolabeled bands (at Mr 84 000 and 60 000
for the dog and at Mr 74 000 and 58 000 for
the pig).
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Autoradiographic Distribution of GHRP Binding Sites in
the Rat Heart
The densitometry analysis of the total binding on four
replicates of rat heart sections gave optical densities of 24.25±2.19
(ODxmm2) (n=4). The optical densities of the
tissue sections relative to the nonspecific binding were 18.00±1.02
(ODxmm2). The difference in the optical
densities from the total and nonspecific binding defined as specific
binding represented 25% of the total signal and was found
to be significant (P<0.05). The specific hexarelin binding
was homogeneously distributed within the heart section as
shown in Figure 9.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is now well-known that GHRPs and peptidomimetics are endowed with strong GH releasing activity in a variety of mammalian species.1 2 3 This effect occurs most likely through interaction with a G protein–coupled receptor cloned recently from porcine, human, and rat pituitary libraries. This 366 amino acid protein of Mr 41 000 with seven transmembrane spanning domains is most likely coupled through activation of G
11 to phosphoinositol
metabolism.5 GHRPs may also interact with the
Mr 57 000 receptor subtype identified in
pituitary membranes.8 Using the Langendorff perfused
heart, we have revealed an unexpected cardiovascular
effect of hexarelin, a hexapeptide member of the GHRP family. Hexarelin
presents a selective dose-response curve of increasing
coronary perfusion pressure in the rat heart. This contrasts
with the dose-response curves obtained with the peptidomimetic
spiroindoline derivative MK-0677 and the shorter analog of GHRP,
EP51389. Given that both compounds are known to be potent GH
secretagogues, there is the possibility of a different
structure-activity profile for GHRP binding sites within the heart. The
signal transduction pathways mediating the cardiac effect of hexarelin
seem to involve L-type Ca2+ channels, because the
increase in coronary perfusion pressure induced by the
hexapeptide is strongly altered by nifedipine. However, the
contribution of other types of Ca2+ channels
could not be excluded. Furthermore, coronary vasoconstriction
in response to hexarelin appears to also involve PKC, because both
chelerythrine and bisindolylmaleimide partially inhibit the
vasoconstrictive effect induced by hexarelin. The
reduced vasoconstriction induced by hexarelin in the presence of the
above-mentioned inhibitors suggests that other transduction
pathways potentially involving tyrosine kinases may be linked with the
vasoconstrictive effect observed.24 The
vasoconstrictive response to hexarelin is probably
endothelium independent, because the
inhibitors of prostaglandin and
thromboxane synthesis, diclofenac and 1-7 CHI,
respectively, did not affect the increase in coronary perfusion
pressure. Hexarelin does not display any chronotropic effect, as heart
rate remained unchanged throughout the perfusion study. However, both
dP/dtmax, an index of myocardial
contractility, and dP/dtmin,
which reflects the ability of the heart to relax, were reduced at the
highest concentration investigated. Although we cannot rule out a
direct action of hexarelin on cardiomyocytes, this effect
is most probably caused by the strong vasoconstriction itself, which
can impair myocardial perfusion and result in myocardial
hypoxia. These results suggest that the effect of hexarelin
might be mediated through GHRP binding sites on vascular smooth muscle
cells. To characterize these binding sites, attempts at equilibrium
binding using the radiolabeled hexarelin derivative
[125I]Tyr-Ala-hexarelin were unsuccessful
because of the hydrophobicity of the GHRP derivative that contributes
to the high level of nonspecific binding.25 26 The
covalent photolabeling approach with benzophenone as the photoreactive
group featuring higher stability than other diazoaryl or arylazide
derivatives27 has allowed the identification of a new
class of GHRP binding sites in the rat heart. Effectively, the results
of the saturation binding assays have shown that the photoactivatable
hexarelin derivative binds to the cardiac membrane protein at
Mr 84 000 with a high affinity. However,
according to the results of competition binding curves, the majority of
these binding sites seem to have a lower affinity for hexarelin, given
that the ED50 value of hexarelin to displace the
photoactivatable ligand was found to be in the micromolar range. The rank order of potencies of GHRP derivatives to reduce the binding of the photoactivatable hexarelin derivative is well correlated with the rank order of potencies of these ligands in increasing the coronary perfusion pressure in the Langendorff perfused heart system. The difference in ED50 values of the peptidyl and nonpeptidyl derivatives of hexarelin, EP51389 and MK-0677, in competing with photoactivatable hexarelin derivative for binding to cardiac and pituitary membranes8 suggests that the cardiac GHRP binding sites are distinct from those identified in the pituitary. Moreover, these cardiac GHRP binding sites appear to be highly glycosylated, contrasting with pituitary GHRP receptors identified recently by the photolabeling approach. These binding sites are also distinct from vasoactive neuropeptide receptors expressed in the coronary vasculature, because neither NPY, neurotensin, CGRP, nor substance P competes for binding of the photoactivatable ligand. Furthermore, the photoaffinity-labeled signal is not altered by circulating vasoactive hormones such as endothelin 1, angiotensin II, or arginine-vasopressin, for which specific receptors are found in the cardiac vasculature.
The regional distribution of the photoactivatable hexarelin derivative has shown that GHRP binding sites are mostly confined to the ventricles, contrasting with the low density observed in the atria. The autoradiographic distribution of this hexarelin derivative reveals that specific binding is present throughout the rat heart sections. However, the resolution of the technique does not allow the cellular localization of the GHRP binding sites in the rat heart. Considering that significant binding is present throughout the thickness of the heart section, the presence of GHRP binding sites on the microvascular system and/or the cardiomyocytes might be expected.
These cardiac GHRP binding sites are found to be widely distributed among various species. Although a unique form of the GHRP receptor at Mr 84 000 was detected in the cardiac membranes from rodents, two forms of the receptor are expressed in membranes from dog and porcine hearts, which most probably reflect different degrees of glycosylation. The ability of endoglycosidase F to reduce the apparent molecular mass of the photolabeled cardiac GHRP receptor from Mr 84 000 to 57 000 suggests that this receptor contains N-linked oligosaccharides. The difference in sensitivity of the cardiac GHRP receptor to N-glycosidase F compared with that from the pituitary indicates a distinct glycosylation pattern of this protein.
Recent studies have demonstrated the cardiovascular effect of GH and its local effector IGF-1 through the interaction with specific receptors expressed at the myocardial level.11 12 GH and IGF-1 have been found to play a critical role in cardiac remodeling and inotropism.28 29 However, hexarelin, a GHRP derivative, appears to have a direct effect on vascular tone, contrasting with the pharmacological profile of GH and IGF-1.
In conclusion, we reported for the first time the existence of specific GHRP binding sites in the mammalian heart, using a photoactivatable hexarelin derivative. We demonstrated that these cardiac GHRP receptors are distinct from the pituitary GHRP receptors involved in GH secretion. Hexarelin appeared to bind with low affinity to these cardiac receptors, inducing potent but reversible coronary vasoconstriction in the perfused Langendorff rat heart. Our results suggest the putative existence of a specific peptidic hormone as endogenous ligand for the cardiac GHRP receptor characterized. This new hormone may be involved in the regulation of vascular tone within the heart.
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Acknowledgments |
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This work was supported by Pharmacia-Upjohn and the Medical Research Council of Canada (PMAC-MRC program). We thank Dr Romano Deghenghi for his generous gift of MK-0677 and EP51389 and Dr Andre De Lean for the access to autoradiographic facilities.
Received July 2, 1999; accepted August 19, 1999.
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