However ghrelin has a short

However, ghrelin has a short half-life of 15–20 min, because it is a natural peptide containing 28-amino acids, its clinical usefulness as a therapeutic agent may be limited (Mosińska et al., 2017; Vestergaard et al., 2007). Hence, many efforts have been made to elucidate the smallest segment of ghrelin which can still activate GHS-R1α (Bednarek et al., 2000; Matsumoto et al., 2001). Structure-activity studies on ghrelin sequence showed that the modification of octanoylation at serine 3 residue is essential for ghrelin’s biological activities (Dehlin et al., 2008; Sato et al., 2012). The amino Cystamine dihydrochloride sequences of ghrelin are well conserved across species, with the 10 amino acids at the N-terminus of ghrelin being identical in humans, pigs, cows, sheep, cats and dogs (Kojima and Kangawa, 2002). Through the systematic studies on the structure-activity relationship of ghrelin molecules, it has been identified that the N-terminal penta-peptide ghrelin(1–5)-NH2 is the minimal active sequence with equipotent activity to the full-length ghrelin as agonist at GHS-R1α (Bednarek et al., 2000; Matsumoto et al., 2001) (Table 1). Furthermore, ghrelin(1–6)-NH2, ghrelin(1–7)-NH2 and ghrelin(1–9)-NH2 have better intracellular calcium-increasing activities than ghrelin(1–5)-NH2. Ghrelin(1–7)-NH2 has the best activity among the all active segments of ghrelin (Matsumoto et al., 2001) (Table 1). All the active segments are the agonists of GHS-R1α. Although ghrelin(1–7)-NH2 is the agonist of GHS-R1α with good activity like ghrelin (Matsumoto et al., 2001), however, there is little knowledge about the biological role of ghrelin(1–7)-NH2. Specifically, there is no research about the effects of ghrelin(1–7)-NH2 on the acute heat pain at the supraspinal level. The distribution sites after i.c.v. injection of ghrelin(1–7)-NH2 need to be further examined in mice. Furthermore, the molecular mechanisms involved in antinociceptive effects induced by ghrelin(1–7)-NH2 (i.c.v.) need to be examined deeply. It has been shown that there is a tight relationship between the ghrelin system and the opioid system, and ghrelin(1–7)-NH2 is the active segment of ghrelin. Therefore, the aim of this study was to (1) examine the roles of i.c.v. injection of ghrelin(1–7)-NH2 on the acute heat pain using the tail withdrawal test in mice, (2) investigate the underlying receptor mechanism(s) using various opioid antagonists and GHS-R1α antagonist [D-Lys3]-GHRP-6, (3) explore the molecular mechanisms of ghrelin(1–7)-NH2 (i.c.v.) using quantitative RT-PCR and Western blot in mice, and (4) explore the distribution sites after i.c.v. injection of ghrelin(1–7)-NH2 in mice using the technologies of fluorescence labeling.
Materials and methods
Results
Discussion The growth hormone secretagogue receptor (GHSR) was cloned in 1996 which is a member of G-protein coupled receptors (Howard et al., 1996), and 3 years later, ghrelin was discovered as its endogenous ligand (Kojima et al., 1999). Ghrelin displays several physiological and pathological functions through binding to GHS-R1α. In humans, rats and mice, the ghrelin genes are located on chromosome 3, chromosome 4 and chromosome 6, respectively. After the transcription and translation of the ghrelin gene, nascent ghrelin peptides are subjected to a unique posttranslational modification: acylation of the hydroxyl group of the Ser3 with n-octanoyl group (Nishi et al., 2011). Then, this ghrelin precursor peptide in which the serine 3 residue is n-octanoylated is converted to the mature 28-amino acid ghrelin peptide by the prohormone convertase PC1/3 (Zhu et al., 2006). The n-octanoyl group modification of Ser3 is essential for ghrelin’s biological activities (Sato et al., 2012). Systematic studies on the structure-activity relationship of ghrelin molecules identified that the N-terminal heptapeptide ghrelin(1–7)-NH2 is the best active sequence of ghrelin. Furthermore, ghrelin(1–7)-NH2 is an agonist of GHS-R1α (Matsumoto et al., 2001). However, there is no research about the effects of ghrelin(1–7)-NH2 on the acute heat pain at the supraspinal level.