Cardiac effects of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln14-ghrelin

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Abstract

The mechanisms underlying the cardiac activities of synthetic growth hormone secretagogues (GHS) are still unclear. The natural ligand of the GHS receptors, i.e. ghrelin, classically binds the GHS receptor and exerts endocrine actions in acylated forms only; its cardiovascular actions still need to be investigated further. In order to clarify these aspects, we studied the effects of either the synthetic peptidyl GHS hexarelin (1 μM), or the natural ghrelin (50 nM) and the endogenous ghrelin derivatives des-Gln14-ghrelin (1–100 nM) and des-octanoyl ghrelin (50 nM), on the tension developed by guinea pig papillary muscle and on L-type Ca2+ current (ICa) of isolated ventricular cells. The binding of these molecules to ventricular cell membrane homogenates was also studied. We observed that all peptides reduced the tension developed at low frequencies (60–120 beats/min) in a dose-dependent manner. No alteration in cardiac contractility was induced by des-Gln14-ghrelin or des-octanoylated ghrelin when the endocardial endothelium had been removed or after cyclooxygenase blockade. Pretreatment with tyramine (2 μM) had no effect on the inotropic response induced by des-Gln14-ghrelin. No significant effect on ICa of isolated ventricular cells was observed in the presence of des-Gln14-ghrelin (100 nM). The order of potency on the tension of papillary muscle was: des-octanoyl ghrelin>ghrelin=des-Gln14-ghrelin>hexarelin. This gradient of potency was consistent with the binding experiments performed on ventricular membranes where either acylated or unacylated ghrelin forms, and hexarelin, recognized a common high-affinity binding site. In conclusion, ghrelin, des-Gln14-ghrelin and des-octanoyl ghrelin, show similar negative inotropic effect on papillary muscle; as des-octanoyl ghrelin is peculiarly devoid of any GH-releasing activity, the cardiotropic action of these molecules is independent of GH release. The binding studies and the experiments performed both on the isolated cells and on papillary muscle after endothelium removal or cyclooxygenase blockade indicate that the cardiotropic action of natural and synthetic ghrelin analogues reflects the interaction with a novel GHS receptor (peculiarly common for ghrelin and des-octanoyl ghrelin), leading to release of cyclooxygenase metabolites from endothelial cells, as indicated by direct measurement of prostacyclin metabolite 6-keto-PGF.

Introduction

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) influence myocardial morphology and functions acting on specific cardiac receptors (Colao et al., 2001). GH and IGF-1 excess or deficit are generally associated with deranged myocardial structure and performance Broglio et al., 1999, Osterziel et al., 2000.

Cardiotropic activities are also exerted by other molecules known as GH secretagogues (GHS) that are distinct entities from GHRH, the endogenous hypophysiotropic neurohormone stimulating somatotroph secretion Ghigo et al., 1997, Müller et al., 1999. GHS were discovered as a family of non-natural peptidyl and non-peptidyl molecules able to strongly stimulate GH secretion acting on a specific G-protein coupled receptor, namely GHS type 1a receptor, that is different from the GHRH-receptor Smith et al., 1997, Muccioli et al., 1998, Muccioli et al., 2002. Theoretically, GHS would therefore influence cardiac structure and function via enhanced GH and IGF-I secretion. However, it has also been demonstrated that these molecules, at least the peptidyl ones, exert also direct and GH-independent cardiac actions. This is suggested by the observations that these effects occur also in hypopituitaric patients (Muccioli et al., 2000) and that are not exerted by GHS analogues (i.e. EP-51389) with strong GH-releasing activity, but lacking cardiac binding sites Locatelli et al., 1999, Müller et al., 2002. In particular, peptidyl GHS, such as hexarelin, improve the post-ischemic recovery of hearts of either aged or GH-deficient rats (Locatelli et al., 1999) and increase left ventricular ejection in patients with severe GH-deficiency Bisi et al., 1999, Imazio et al., 2002. A stimulatory effect on cell proliferation (Pettersson et al., 2002) and an antiapoptotic action (Filigheddu et al., 2001) of GHS on the cardiomyocyte cell line H9c2 have also been shown. The natural ligand of the GHS type 1a receptor has more recently been discovered as a gastric-derived hormone named ghrelin (Kojima et al., 1999). It is an acyl-peptide consisting of 28 amino acids and esterified with octanoic acid on Ser3, though a form of 27 amino acids, des-Gln14-ghrelin (resulting from alternative splicing of the same gene) has also been isolated (Hosoda et al., 2000a). Both exist in either acylated and, more abundantly, non-acylated form (Hosoda et al., 2000b), but the latter have been shown inactive in term of endocrine activities (Kojima et al., 2001). They do not stimulate GH secretion in vivo (Torsello et al., 2002) because do not bind and activate the GHS type 1a receptor (Bednarek et al., 2001) neither displace radiolabelled ghrelin from its hypothalamo-pituitary binding sites Muccioli et al., 2001, Torsello et al., 2002. The gastric peptide ghrelin is endowed with a strong stimulatory effect on GH release, gastric acid secretion and gut motility in rats and humans (Kojima et al., 2001). However, many recent studies have demonstrated that ghrelin is much more than a natural GH secretagogue and that its biological actions are not confined to the pituitary or to the gastro-intestinal tract. In fact, it has been reported that ghrelin increases food intake, adiposity and causes a positive energy balance, as well as modulates glucose metabolism and cell growth in non-tumoural and neoplastic tissues (Muccioli et al., 2002). In addition, as a result of studies conducted in vitro and in vivo, it has been already reported that ghrelin not only exerts cardiovascular effects in humans Muccioli et al., 2000, Okumura et al., 2002, Nagaya and Kangawa, 2003, but it has also been demonstrated that ghrelin does not share all cardiac activities exerted by synthetic GHS (Torsello et al., 2003). Interestingly, besides GHS type 1a receptor mRNA expression (Nagaya and Kangawa, 2003) and specific binding sites labelled by [125I]His9-ghrelin (Katugampola et al., 2001), animal and human myocardium possess also GHS receptors that are specific for peptidyl GHS and which do not recognise ghrelin or the non-peptidyl GHS, MK-0677 Bodart et al., 1999, Papotti et al., 2000. The recent finding that H9c2 cardiomyocytes do not express GHS type 1a receptor, but have high affinity binding sites, common for ghrelin and des-acyl ghrelin, involved in mediating their antiapoptotic activity, provides further support to the hypothesis that multiple ghrelin and GHS receptors exist in the cardiovascular system (Baldanzi et al., 2002). Each receptor may then contribute independently to the wide array of cardiovascular activities induced by synthetic GHS, ghrelin and endogenous ghrelin-derived molecules Muccioli et al., 2000, Muccioli et al., 2002, Torsello et al., 2003.

In order to clarify the mechanisms underlying the cardiac activities of ghrelin and to verify whether its different molecular forms are cardioactive or not, we have studied the effects of ghrelin (50 nM), des-Gln14-ghrelin (1–100 nM) and des-octanoyl ghrelin (50 nM), as well as of the synthetic peptidyl GHS hexarelin (1 μM), on the tension developed by guinea pig papillary muscle, a model that shows a biphasic force–frequency relationship similar to that reported for human cardiac muscle (Brixius et al., 1999). The effects of ghrelin and its derivatives on L-type calcium current (ICa) of isolated ventricular cells and the binding of all these molecules to ventricular cell membrane homogenates have also been studied.

Section snippets

Materials

Human ghrelin (Gly-Ser-Ser-(O n-octanoyl)-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg-NH2) and des-octanoyl ghrelin were purchased from Phoenix Pharmaceuticals (Belmont CA, USA), whereas des-Gln14-ghrelin was both from Tocris (Pallwin, MO, USA) and Phoenix Pharmaceuticals, Tyr4-ghrelin, Tyr4-des-octanoyl ghrelin, and hexarelin (His-d-2Me-Trp-Ala-Trp-d-Phe-Lys-NH2) were purchased from Neosystem (Strasbourg, France). [125I]Tyr4-ghrelin

Contractile effect on isolated papillary muscle

In previous experiments, we observed that hexarelin modifies the force–frequency relationship in rat papillary muscle, reducing the tension developed at low frequencies, while at higher frequencies contractile force was unaltered (Bedendi et al., 2001). We observed that, similarly to the rat, also in the guinea pig papillary muscle hexarelin (1 μM) reduces the tension developed at low frequencies (60–120 beats/min), while the tension at higher frequencies (150–320 beats/min) is unaffected (Fig.

Discussion

The results of present study demonstrate that ghrelin, des-Gln14-ghrelin or des-octanoyl ghrelin, as well as hexarelin, a synthetic peptidyl growth hormone secretagogue (GHS), all show similar negative inotropic effect on the guinea pig papillary muscle. Interestingly, the most significant influence on contractility of the papillary muscle was exerted by des-octanoyl ghrelin, an unacylated form devoid of any endocrine action Kojima et al., 2001, Torsello et al., 2002 because this form, unlike

Acknowledgments

The authors wish to thank Prof. F. De Matteis (University of Turin) for suggestions and for critically reviewing the manuscript. This work was supported by grants (ex-60% 2001 to G.M. Cofin 2000 to G.A. G.M., E.G. and Cofin 2002 to G.M.) from the Italian Ministry of University and Research, Rome and by grants from INFM, Compagnia di San Paolo di Torino and the Fondazione per lo Studio delle Malattie Endocrine e Metaboliche (SMEM Foundation, Turin, Italy).

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