Regionally-dependent inhibition or facilitation of cholinergically-mediated contractions by motilin in human distal esophagus and gastric fundus

John Broad1, Mahsa Samedi2, George Dukes3, Gareth Sanger1. 1Neurogastroenterology group, Blizard Institute, Barts & The London School of Medicine and Dentistry, Queen Mary University of London, London, UK, 2Centre for Integrative Physiology, University of Edinburgh, Edinburgh, UK, 3Academic DPU, GlaxoSmithKline, 3030 E. Cornwallis Road, Research Triangle Park, NC 27709,, Durham, USA

The gastrointestinal hormone motilin is released during hunger to mediate phase III of the migrating motor complex and when administered after meals, stimulates gastric emptying (Sanger 2008). Surprisingly, atilmotin, a motilin-derived peptide, reduced esophageal contractions in healthy volunteers whilst contracting the lower esophageal sphincter and stomach (Korimilli & Parkman 2010). Regionally-dependent neuromuscular actions of motilin have now been looked for using human isolated esophagus and stomach.

Distal esophagus and gastric fundus were obtained at surgery for cancer (macroscopically normal areas) or obesity, with informed consent. After removing the mucosa, strips (3–5 x 15 mm) were cut parallel to the circular muscle and suspended between platinum ring electrodes in baths (Krebs’; 37°C; 5% CO2 in O2; 2g tension; isometric recording) for electrical field stimulation (EFS; 50V, 0.5 ms bipolar pulses, 1-20 Hz, given for 10 s every 1 min). Separately, contractions were evoked by carbachol 1µM (5 min contact, 15-20 min intervals). Motilin was applied non-cumulatively. Formalin-fixed sections were stained using a motilin receptor antibody (1:1200, MBL international). N = number of patients.

Motilin receptor immunoreactivity was observed in the muscle layers and myenteric plexus of the esophagus and fundus. In esophagus, EFS 1-20Hz evoked frequency-dependent contractions in most strips, with a second ‘after-contraction’ on termination of EFS (48/52; n = 18). In fundus, the evoked contractions were monophasic and frequency-dependent (110 strips; n = 34). Subsequently, only 5Hz was used. All responses were prevented by 1µM tetrodotoxin or 1µM atropine (n = 3-5 each). L-NAME 300µM increased contractions during EFS (esophagus 239±59%, n = 9; fundus 57±17%, n = 15) and abolished after-contractions in the esophagus. Motilin 1-300nM had no effects on muscle tension in the esophagus but 10-300nM caused short-lived inhibition of contractions during EFS (pIC50 = 7.2; Imax = 47±26%, t½ = 13±5 min; n = 3-5), an activity unaffected by L-NAME 300µM (30 min pre-incubation; 31±7% inhibition at 300nM, n = 6) and not observed when carbachol was used to contract the muscle. In the fundus, motilin increased muscle tension (e.g. by 124±27% of the pre-drug contraction amplitudes to EFS at 300nM) and facilitated contractions evoked by EFS (pEC50 = 7.0; Emax 82±42%, t½ 17±5 min; n = 3-5).

The cholinergic and nitrergic phenotypes of responses to EFS in human distal esophagus and fundus are consistent with previous reports (Richards et al 1995; Broad et al 2010). Motilin acted prejunctionally to decrease cholinergically-mediated contractions in esophagus and increase contractions in the fundus; activity in the esophagus was independent of nitric oxide.

Broad J et al (2010). Neurogastroenterol Motil, 22 (S1), 84.

Korimilli A, Parkman HP (2010). Dig Dis Sci, 55, 300-306.

Richards WG et al (1995). J Thoraxic Cardiovasc Surg, 110, 157-164.

Sanger GJ (2008). Drug Discovery Today, 13, 234-239