Ghrelin increases electrically-evoked, neuronally-mediated contractions in mouse (Murray et al., 2002) and rat (Dass et al., 2003) isolated forestomach. This activity is associated with ghrelin receptor immunoreactivity in the enteric nervous system (Dass et al., 2003) and an ability of ghrelin to increase gastric emptying (eg. Trudel et al., 2002). The enteric nerve phenotype sensitive to ghrelin is unknown so we re-examined the activity of ghrelin in vitro, in the presence of antagonists at the receptors for the main gastric motor neurotransmitters.

Methods: Circular muscle preparations of rat gastric forestomach were suspended between 2 platinum ring electrodes in Krebs solution (5% CO₂ 95% O₂; pH 7.4; 37^oC) under 10 mN tension for isometric recording. Responses were evoked using electrical field stimulation (EFS) applied for 30 s at 150 s intervals for 30 min periods at 5 Hz (0.5 ms; ±50 V). Responses could be prevented by tetrodotoxin 1 µM (n=8).

Results: EFS evoked a small initial contraction, followed by a further contraction or more usually, by muscle relaxation (57 % of preparations from 54 rats). At termination of EFS, a large rapidly-developing after-contraction was observed. Atropine 1 µM prevented the contractions during EFS and increased any relaxations observed during EFS by 103 ± 24 % (n=9). Atropine also changed the kinetics of the after-contractions, which were now slower to fade and either reduced (3/9 preparations) or increased (6/9) in amplitude compared with the original contraction (overall increase of 55 ± 35 %; P>0.1).

L-NAME 0.3 mM facilitated all contractions and abolished EFS-evoked relaxations replacing these by a contraction (n=5). The neurokinin (NK) NK_1,2,3 receptor antagonists L-732,138 1 µM, MDL-29,913 1 µM and SB-235375 0.1 µM, when added together reduced the after-contraction by 71 ± 29 % (n=11) and abolished any relaxation which occurred during EFS, replacing this with a contraction (n=8). The combination of atropine + NK receptor antagonists usually abolished the after-contractions (n=8). In control tissues, rat ghrelin 1 µM (maximally-effective; Dass et al., 2003) increased the amplitude of the maximum contraction during EFS by 40 ± 22 % (n=9) and reduced EFS-evoked relaxations by 12 ± 4 % (n=6). Ghrelin also increased the after-contractions by 22 ± 7 % (n=14). In the presence of atropine 1 µM, ghrelin 1 µM had no effects on EFS-evoked relaxations and increased the amplitude of the after-contractions by 31 ± 19 % (n=5). In the presence of the NK receptor antagonists (as above), ghrelin 1 µM increased the amplitude of the contractions evoked both during (by 78 ± 25 %; n=7) and after EFS (by 43 ± 15 %; n=7). The effects of ghrelin in the presence of atropine + NK receptor antagonists were not determined as EFS-evoked after-contractions could not be consistently detected.

Conclusions: EFS evokes responses mediated predominantly by cholinergic, nitregic and tachykinergic systems. Ghrelin exerts gastric prokinetic activity by acting on both cholinergic and tachykinergic excitatory pathways.

Dass, N.B., et al., (2003) Neuroscience, 120, 443-453.
Murray, C., et al., (2002) Br. J. Pharmacol., 136, 18P.
Trudel, L., et al., (2002) Am. J. Physiol., 282, G948-G952.