As for the experiments with motilin (see above), the potency of GSK962040 was lower than when tested at the recombinant human receptor (pEC50 values of 7.9 and 4.8 respectively; see Sanger et al., 2009), perhaps explained by the additional need to penetrate into the isolated tissue. immunoreactivity was identified in muscle and myenteric plexus predominantly in the upper gut, co-expressed with choline acetyltransferase in neurons. CONCLUSIONS AND IMPLICATIONS Motilin and GSK962040 strongly facilitated cholinergic activity in the antrum, with lower activity in fundus and small intestine only. Facilitation by motilin was short lived, consistent with participation in migrating motor complexes. Long-lasting facilitation by GSK962040 suggests different receptor interactions and potential for clinical evaluation. LINKED ARTICLE This article is usually commented on by Depoortere, pp. 760C762 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02046.x values are numbers of patients. Differences between medians were decided using the MannCWhitney < 0.05 was considered statistically significant. Materials All drugs were freshly prepared before use. Motilin (Tocris, Abingdon, UK) was dissolved in distilled water at 100 M. GSK962040 R112 ((N-(3-fluorophenyl)-1-[(4-[(3S)-3-methyl-1-piperazinyl]methylphenyl)acetyl]-4-piperidinamine) was dissolved in distilled water to 1 1 Rabbit Polyclonal to CDK8 mM. Carbachol, atropine, N-nitro-L-arginine methyl ester hydrochloride (L-NAME; Sigma) and tetrodotoxin (TTX) (Tocris, UK) were each dissolved in distilled water. L732138 (Tocris) was dissolved in DMSO (Sigma) to 10 mM; “type”:”entrez-nucleotide”,”attrs”:”text”:”GR159897″,”term_id”:”238420493″,”term_text”:”GR159897″GR159897 (Tocris) and SB235375 (GSK) were dissolved in ethanol to 10 mM. Stock solutions of captopril and phosphoramidon (10 mM) and of thiorphan (5 mM) were prepared in distilled water and of chymostatin (2.5 mM) in DMSO (Sigma). Results Motilin receptor immunoreactivity Motilin receptor immunoreactivity was identified in all regions of stomach and intestine, particularly the upper gut. Punctate labelling was observed in longitudinal and circular muscle, and in the myenteric plexus (Physique 1). In the myenteric plexus this was detected mostly as punctate labelling of neuronal cell body membranes, with some additional immunoreactivity within the cytosol. Not all neuronal cells were labelled and immunoreactivity was not detected in nerve axons labelled by neurofilament 2F11, either in the myenteric R112 plexus or the muscle layers of the antrum (data not shown). Open in a separate window Physique 1 Motilin receptor immunoreactivity in R112 the human gastrointestinal tract. The upper panel (A; immunohistochemical studies) illustrates the distribution of motilin receptors (stained in red) to the longitudinal and circular muscle layers of the antrum, and to the myenteric plexus. Illustrations are 10 R112 and 25. Ganglion cells of the plexus show some granular peripheral staining for the receptor (examples highlighted by arrows). Staining was not observed with the unfavorable control (no primary antibody; not shown). Panel (B) shows motilin receptor immunoreactivity in gastric fundus, duodenum, terminal ileum and colon. Illustrations are 25 magnification. Panel (C; immunofluorescence studies) shows an expanded view of the co-localization of motilin receptors with ChAT immunoreactivity in the antrum. LM = longitudinal muscle, CM = circular muscle, MP = myenteric plexus. Within the myenteric plexus of the stomach, motilin receptor immunoreactivity was co-localized with choline acetyltransferase (ChAT) (Physique 1). Of the 53 cells positive for ChAT in the antrum, 35 (66%) also expressed motilin receptors R112 (7 ganglia, < 0.01 compared with tissues used on day of surgeryStomach (25)Esophagogastric cancer1:0.563 (40C87)Small and large intestines (23)Ileo-colonic resection for cancer1:1.366 (18C88) Open in a separate window (strips)> 0.05]. Analysis of contractions to EFS (5 Hz) under different baseline tensions showed that responses reached maximum at around 2 g in both fundus and antrum, so 2 g was used for all tissues (1.20 0.33, 1.43 0.75, 1.36 0.34 g (g wet weight)?1 was the tension developed under 2 g load in, respectively, antrum from obese patients, fundus from obese patients and fundus from cancer patients, > 0.05 each; > 0.5). Interestingly, at 300 nM motilin, the decline in contractile amplitude did.
As for the experiments with motilin (see above), the potency of GSK962040 was lower than when tested at the recombinant human receptor (pEC50 values of 7