Identification of a Pituitary Growth Hormone-Releasing Peptide (GHRP) Receptor Subtype by Photoaffinity Labeling

143

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 125 I-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 125 I-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 (K d ) of (1·5 0·3) 10 9 and (2·1 0·4) 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 125 I-labelled Tyr-Ala-hexarelin. The binding of 125 Ilabelled 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 [-Arg 1 --Phe 5 --Trp 7,9 -Leu 11 ]-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.

Section: Chemicalssupporting

confidence: 66%

Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland

Muccioli

Ghè²,

Ghigo³

et al. 1998

143

“…Furthermore, it has been suggested that different subtypes of the recently cloned GHS receptor may exist and that different GHSs may differ in their binding affinity for the different subtypes (Ong et al 1998). Despite these differences, the effects on body growth and BMC were similar for all treatments with the doses and the route of administration used in the present study.…”

Section: Tablementioning

confidence: 52%

The GH secretagogues ipamorelin and GH-releasing peptide-6 increase bone mineral content in adult female rats

Svensson

Lall²,

Dickson³

et al. 2000

Growth hormone (GH) is of importance for normal bone remodelling. A recent clinical study demonstrated that MK-677, a member of a class of GH secretagogues (GHSs), increases serum concentrations of biochemical markers of bone formation and bone resorption. The aim of the present study was to investigate whether the GHSs, ipamorelin (IPA) and GH-releasing peptide-6 (GHRP-6), increase bone mineral content (BMC) in young adult female rats. Thirteen-week-old female Sprague-Dawley rats were given IPA (0·5 mg/kg per day; n=7), GHRP-6 (0·5 mg/kg per day; n=8), GH (3·5 mg/kg per day; n=7), or vehicle administered continuously s.c. via osmotic minipumps for 12 weeks. The animals were followed in vivo by dual X-ray absorptiometry (DXA) measurements every 4th week. After the animals were killed, femurs were analysed in vitro by mid-diaphyseal peripheral quantitative computed tomography (pQCT) scans. After this, excised femurs and vertebrae L6 were analysed by the use of Archimedes' principle and by determinations of ash weights. All treatments increased body weight and total tibial and vertebral BMC measured by DXA in vivo compared with vehicle-treated controls. However, total BMC corrected for the increase in body weight (total BMC:body weight ratio) was unaffected. Tibial area bone mineral density (BMD, BMC/area) was increased, but total and vertebral area BMDs were unchanged. The pQCT measurements in vitro revealed that the increase in the cortical BMC was due to an increased cross-sectional bone area, whereas the cortical volumetric BMD was unchanged. Femur and vertebra L6 volumes were increased but no effect was seen on the volumetric BMDs as measured by Archimedes' principle. Ash weight was increased by all treatments, but the mineral concentration was unchanged. We conclude that treatment of adult female rats with the GHSs ipamorelin and GHRP-6 increases BMC as measured by DXA in vivo. The results of in vitro measurements using pQCT and Archimedes' principle, in addition to ash weight determinations, show that the increases in cortical and total BMC were due to an increased growth of the bones with increased bone dimensions, whereas the volumetric BMD was unchanged.

“…Recent studies have demonstrated other subtypes of the receptor, which are expressed also in peripheral tissues (McKee et al 1997b, Tan et al 1998. Specific binding sites for GHSs have been found in thyroid and cardiac (Ong et al 1998a, Bodart et al 1999, 2002 tissue.…”

Section: Introductionmentioning

confidence: 99%

“…Specific binding to rat cardiac membranes has been suggested using an iodinated and photoactivatable derivative of hexarelin, 125 I-labeled Tyr-Bpa-Ala-hexarelin (Ong et al 1998a, Bodart et al 1999. Papotti et al (2000) have also shown specific binding to human cardiac tissue using iodinated Tyr-Ala-hexarelin.…”

Section: Introductionmentioning

confidence: 99%

Natural (ghrelin) and synthetic (hexarelin) GH secretagogues stimulate H9c2 cardiomyocyte cell proliferation

Pettersson

Muccioli²,

Granata³

et al. 2002

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 µM (147 3% of control, P<0·01) and elicited maximal effects at concentrations around 30 µM. 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 µM 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-Alahexarelin 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 µM 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.