Recent experimental data demonstrate cardiovascular effects of the GH secretagogues (GHSs) hexarelin and ghrelin, the proposed natural ligand for the GHS receptor. Moreover, specific cardiac binding sites for GHSs have been suggested. The aim of the present study was to investigate if the natural ligand ghrelin and synthetic GHS peptide hexarelin and analogues have direct effects on the cardiomyocyte cell line, H9c2. Hexarelin stimulated thymidine incorporation in a dose-dependent manner with significant responses at 3 micro M (147+/-3% of control, P<0.01) and elicited maximal effects at concentrations around 30 micro M. This activity was seen already after 12 h of incubation with a maximal effect after 18 h (176+/-9% of control, P<0.01). Ghrelin also had a significant stimulatory effect on thymidine incorporation (129+/-2% of control at 3 micro M and 18 h, P<0.05). The stimulatory effect on thymidine incorporation of hexarelin, Tyr-Ala-hexarelin, EP80317 and ghrelin was specific and no stimulatory effect was observed with the truncated GH-releasing peptide EP51389 or the non-peptidyl GHS MK-0677. In competitive binding studies, (125)I-labeled Tyr-Ala-hexarelin was used as radioligand and competition curves showed displacement with hexarelin, Tyr-Ala-hexarelin, EP80317 and ghrelin, whereas MK-0677 and EP51389 produced very little displacement at 1 micro M concentration, adding further support for an alternative subtype binding site in the heart compared with the pituitary. In conclusion, we have demonstrated a dose-dependent and specific stimulation of cardiomyocyte thymidine incorporation by natural and synthetic GHS analogues, suggesting increased cell proliferation and binding of GHS to H9c2 cardiomyocyte cell membranes. These findings support potential peripheral effects of GHS on the cardiovascular system independent of an increased GH secretion.

The presence of specific receptors for synthetic growth hormone secretagogues (GHSs) has been investigated in non-tumoral and neoplastic human thyroid tissue using a radio-iodinated peptidyl GHS ((125)I-labelled Tyr-Ala-hexarelin) as ligand. Specific binding sites for Tyr-Ala-hexarelin were detected in membranes from non-tumoral and follicular-derived neoplastic thyroid tissue, but not in thyroid tumours (medullary carcinomas) of parafollicular (C cell) origin. The binding activity was greatest in well differentiated neoplasms (papillary and follicular carcinomas), followed by poorly differentiated carcinomas, non-tumoral thyroid parenchyma, follicular adenomas and anaplastic carcinomas. Both peptidyl (Tyr-Ala-hexarelin, hexarelin, growth hormone releasing peptide (GHRP6) and non-peptidyl (MK 0677) GHSs completely displaced the radioligand from binding sites of non-tumoral thyroid gland, but MK 0677 was significantly less potent. The IC(50) values were (1. 9+/-0.3)x10(-8) mol/l for Tyr-Ala-hexarelin, (2.1+/-0.2)x10(-8) mol/l for hexarelin, (2.4+/- 0.3)x10(-8) mol/l for GHRP6 and only (1. 5+/-0.4)x 10(-7) mol/l for MK 0677. Similar IC(50) values were found in neoplastic thyroid tissue. Scatchard analysis of the binding revealed a finite number of binding sites in non-tumoral (B(max): 1232+/-32 fmol/mg protein, n=3) and neoplastic (papillary carcinomas) thyroid tissue (B(max): 2483+/-380 fmol/mg protein, n=5), with dissociation constants (K(d)) of (3.8+/-0.3)x10(-9) and (4. 4+/-0.6)x 10(-9) mol/l, respectively. On the basis of this evidence, we investigated the effects of some GHS on the proliferation of three different human follicular thyroid carcinoma cell lines (NPA, WRO and ARO) in which the presence of specific GHS receptors was also demonstrated. Tyr-Ala-hexarelin, GHRP6 and MK 0677 were able to inhibit serum-stimulated [(3)H]thymidine incorporation in NPA cells at concentrations close to their binding affinity. These substances also caused a significant inhibition of cell proliferation, which was evident at the earliest time of treatment (24 h) in all the cell lines, and at the latest time (96 h) in NPA cells only. In conclusion, this paper confirms the existence of specific binding sites for GHS in normal thyroid tissue and demonstrates, for the first time, that these binding sites are present in papillary and follicular carcinomas, low in anaplastic carcinomas and absent in medullary carcinomas of the thyroid. This work also provides evidence of a growth-inhibitory effect of GHS on cell lines derived from follicular thyroid cancers.

Abstract

The aim of our study was to investigate the effect of hexarelin, a novel GH-releasing peptide-6 analog, and GH-releasing hormone (GHRH) (alone or in combination) on GH secretion in adult patients with increased somatostatin tone due to chronic glucocorticoid excess. We studied seven adult patients undergoing long-term (no less than 6 months) immunosuppressive glucocorticoid treatment for non-endocrine diseases (six females and one male, age range 42–68 years) and one subject (female, age 31 years) with endogenous hypercortisolism due to adrenal adenoma. Six normal subjects (four females and two males) matched for sex and age with the patients and not undergoing any therapy served as controls. All the subjects underwent the following three tests in random order: (1) human GHRH (1–29)NH₂ (100 μg in 1 ml saline) injected as an i.v. bolus at 0 min, (2) hexarelin (100 μg in 1 ml saline) injected as an i.v. bolus at 0 min and (3) hexarelin (100 μg in 1 ml of saline) plus GHRH (100 μg in 1 ml saline) injected as an i.v. bolus at 0 min. After GHRH alone the patients with glucocorticoid excess showed a blunted GH response as compared with normal subjects (median delta GH: 0·9, range 0–5·6 μg/l vs 7·1, range 0·3–14·9 μg/l). No significant differences were observed in the steroid-treated group with respect to normal subjects after hexarelin alone (median delta GH: 15·5, range 1·9–45·2 μg/l vs 17·9, range 5·5–53·9 μg/l). The GH responses after the combined stimuli were significantly decreased in the patients with glucocorticoid excess as compared with normal subjects (median delta GH: 43·5, range 21–56·9 μg/l vs 73·7, range 19·6-116·8 μg/l). The two stimuli acted in a synergistic fashion in both groups of subjects. It may be hypothesized that hexarelin influences the GH inhibitory effect of glucocorticoids in humans, acting at the hypothalamic somatostatin level.

Journal of Endocrinology (1995) 146, 227–232

In vitro studies have been performed to demonstrate and characterize specific binding sites for synthetic GH secretagogues (sGHS) on membranes from pituitary gland and different human brain regions. A binding assay for sGHS was established using a peptidyl sGHS (Tyr-Ala-hexarelin) which had been radioiodinated to high specific activity at the Tyr residue. Specific binding sites for 125I-labelled Tyr-Ala-hexarelin were detected mainly in membranes isolated from pituitary gland and hypothalamus, but they were also present in other brain areas such as choroid plexus, cerebral cortex, hippocampus and medulla oblongata with no sex-related differences. In contrast, negligible binding was found in the thalamus, striatum, substantia nigra, cerebellum and corpus callosum. The binding of 125I-labelled Tyr-Ala-hexarelin to membrane-binding sites is a saturable and reversible process, depending on incubation time and pH of the buffer. Scatchard analysis of the binding revealed a finite number of binding sites in the hypothalamus and pituitary gland with a dissociation constant (Kd) of (1.5 +/- 0.3) x 10(-9) and (2.1 +/- 0.4) x 10(-9) mol/l respectively. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipids are essential for the binding of 125I-labelled Tyr-Ala-hexarelin. The binding of 125I-labelled Tyr-Ala-hexarelin to pituitary and hypothalamic membranes was displaced in a dose-dependent manner by different unlabelled synthetic peptidyl (Tyr-Ala-hexarelin, GHRP2, hexarelin, GHRP6) and non-peptidyl (MK 0677) sGHS. An inhibition of the specific binding was also observed when binding was performed in the presence of [D-Arg1-D-Phe5-D-Trp7,9-Leu11]-substance P, a substance P antagonist that has been found to inhibit GH release in response to sGHS. In contrast, no competition was observed in the presence of other neuropeptides (GHRH, somatostatin, galanin or Met-enkephalin) which have a known influence on GH release. In conclusion, the present data demonstrate that sGHS have specific receptors in human brain and pituitary gland and reinforce the hypothesis that these compounds could be the synthetic counterpart of an endogenous GH secretagogue involved in the neuroendocrine control of GH secretion and possibly in other central activities.