Ghrelin receptor expression and activities in the mouse gastrointestinal tract

Navjeet Jolly1, Robert Tolhurst1, Annette Beck-Sickinger2, Thue Schwartz3, Helen Cox1. 1Wolfson CARD, King’s College London, London, SE1 1UL, United Kingdom, 2Institute of Biochemistry, University of Leipzig, Germany, 04103, Germany, 3Laboratory for Molecular Pharmacology, The Panum Institute, University of Copenhagen, Denmark, 2200, Denmark.

Ghrelin is the only hunger inducing peptide, produced mainly in the stomach, with promotile effects in mammalian intestine (Chen et al., 2009). It mediates its effects through the ghrelin receptor (GHSR) once acylated by the enzyme ghrelin O-acyltransferase (GOAT). Ghrelin may also activate peripheral receptors in the enteric nervous system and recent studies have documented the presence of the GHSRs in the myenteric plexus (Xu et al., 2005). GHSR exhibits high constitutive activity (Holst et al., 2004a) and therefore drugs that reduce this activity could be useful in the treatment of obesity (Holst et al., 2004b). Our aims were to establish the expression patterns of GHSR, ghrelin and GOAT in the mouse gastrointestinal (GI) tract and to investigate the activity of acyl ghrelin (AG) in isolated GI preparations and upper GI transit in vivo.

We used reverse transcription polymerase chain reaction (RT-PCR) to identify GHSR, ghrelin and GOAT expression along the length of mouse GI tract (adult males, >10wk, C57Bl/6-129/SvJ background), separating mucosal from muscle layers. Changes in isometric smooth muscle tension were measured in tissue from different GI areas in response to AG (1 nM) and carbachol (CCh, 10 µM). The effects of AG (0.1 – 100 nM) on faecal pellet propulsion in isolated full-length colon were determined (pellet movement was expressed as a % of total colon length over 20 min). In vivo upper GI transit was also measured after intra-gastric oral gavage of a charcoal meal (expressed as a % of total intestinal length), 30 min after an i.p. injection of either vehicle (saline, 100 µl) or AG (500 µg/kg, i.p.). Multiple comparisons were performed using one-way ANOVA with a Dunnett’s post-test comparing all groups with the vehicle controls. Individual comparisons were performed using a Student’s t-test.

GHSR expression was highest in the mouse ileum and ascending colon, while ghrelin and GOAT expression were highest in the mucosa and muscle of the fundic area of the stomach. AG (1 nM) had no significant effect on longitudinal muscle tension in isolated tissues from the stomach to descending colon compared with CCh induced contractile responses (which in ascending colon were 1.0 ±0.3g, n = 5). Unexpectedly, in full-length colon we observed significant stasis of faecal pellets with 0.1 nM AG (0.1±0.1%, P<0.001, n = 3), 1 nM AG (1.2 ± 0.3%, P < 0.001, n = 3) and 10 nM AG (3.5 ± 2.2%, P < 0.05, n = 3) compared with vehicle controls (8.2 ± 0.8%, n = 12). AG (500 µg/kg) did however significantly increase SIT (83.6 ± 6.2%, n = 3, P < 0.05) compared with vehicle controls (59.9 ± 1.8%, n = 3).

In conclusion, we demonstrate a comprehensive analysis of GHSR expression along the length of the mouse GI tract. We also corroborate that ghrelin expression decreases along the GI tract and that GOAT expression is restricted mainly to the fundus. We show that AG does not induce any effect on longitudinal smooth muscle tension (in contrast to its promotile effects on circular muscle in the forestomach, Bassil et al., 2005). We demonstrate a consistent inhibition of colonic transit ex vivo with a wide range of AG concentrations, indicating an intramural inhibitory mechanism, possibly at the level of myenteric neurons. Inhibition of enteric reflex activity ex vivo is different from the promotile effects commonly seen for AG in rodent intestine in vivo, but may highlight a localised effect of GHSR activation on inhibitory myenteric motor neurons and/or demonstrate the effect of removal of signalling through the vagus. Finally, we confirm the promotile effect of AG on upper GI transit when administered by i.p. injection.

Bassil et al., (2005) Eur J Pharmacol. 524, 138-144.

Chen et al., (2009) Pharmacol Rev. 61, 430-481.

Holst et al., (2004a) J Biol Chem, 279, 53806-53817.

Holst et al., (2004b) Trends in Pharmacol. Sci, 25, 113-117.

Xu et al., (2005) Regul. Pept. 124, 119–125

Funded by EU 7th Framework Programme, Grant Agreement: No. 223057 (GIPIO). We thank Sarah Forbes for her help with in vivo work and PolyPeptide Group (Hillerød, Denmark) for synthesising AG.