Differential effects of CB(1) neutral antagonists and inverse agonists on gastrointestinal motility in mice

Abstract

Background: Cannabinoid type 1 (CB(1)) receptors are involved in the regulation of gastrointestinal (GI) motility and secretion. Our aim was to characterize the roles of the CB(1) receptor on GI motility and secretion in vitro and in vivo by using different classes of CB(1) receptor antagonists.

Methods: Immunohistochemistry was used to examine the localization of CB(1) receptor in the mouse ileum and colon. Organ bath experiments on mouse ileum and in vivo motility testing comprising upper GI transit, colonic expulsion, and whole gut transit were performed to characterize the effects of the inverse agonist/antagonist AM251 and the neutral antagonist AM4113. As a marker of secretory function we measured short circuit current in vitro using Ussing chambers and stool fluid content in vivo in mouse colon. We also assessed colonic epithelial permeability in vitro using FITC-labeled inulin.

Key results: In vivo, the inverse agonist AM251 increased upper GI transit and whole gut transit, but it had no effect on colonic expulsion. By contrast, the neutral antagonist AM4113 increased upper GI transit, but unexpectedly reduced both colonic expulsion and whole gut transit at high, but not lower doses.

Conclusions & inferences: Cannabinoid type 1 receptors regulate small intestinal and colonic motility, but not GI secretion under physiological conditions. Cannabinoid type 1 inverse agonists and CB(1) neutral antagonists have different effects on intestinal motility. The ability of the neutral antagonist not to affect whole gut transit may be important for the future development of CB(1) receptor antagonists as therapeutic agents.

Figure 1

Representative micrographs of CB₁ immunoreactivity in cross-sections and whole mount preparations of ileum and colon from C57BL/6N male mice. Cross-sections of full thickness ileum (A) and colon (B). Whole mount preparations of the myenteric plexus of the ileum (C), colon (D) and submucosal plexus of the ileum (E) and colon (F). Note the dense innervation of the plexuses and extensive innervation of the mucosa. The epithelium was not labelled. Scale bars: 50μm.

Figure 2

Concentration-dependent effects of (A) AM251, (B) rimonabant, (C) AM4113 (all 1nM-100nM) on electrically-evoked contractions of the mouse ileum and (D) the effects of AM251, rimonabant and AM4113 prior to the addition of WIN55,212-2 on electrically-evoked contractions. Note the significant inhibitory effect of WIN55,212-2 is blocked by the presence of all the CB₁ receptor antagonists. ** p < 0.01; *** p < 0.001; n = 6-9 each point.

Figure 3

(A) AM251 (0.5-1 mg/kg, n=8-12/group) and (B) AM4113 (0.1-1 mg/kg, n=5-6/group) increase upper GI transit of a marker dye in vivo. (C) AM4113 (0.1 mg/kg) and AM251 (0.5 mg/kg) completely block the inhibitory effects of the CB₁ agonist WIN55,212-2 (1 mg/kg, n=4-9/group). Note, the effects of AM4113 (2 mg/kg) and AM251 (1 mg/kg) are absent in the CB₁^-/- mouse (D, n=4-8/group), which has an enhanced upper GI transit compared to wild type control mice. * p<0.05; ** p < 0.01; *** p < 0.001.

Figure 4

(A) AM251 (1-2 mg/kg, n=4-12/group) and (B) AM4113 (1-2 mg/kg, n=6-16/group) have no significant effect on colonic expulsion time in vivo. (C) AM4113 (1 mg/kg) and AM251 (2 mg/kg) completely block the inhibitory effect of the CB₁ agonist WIN55,212-2 (3 mg/kg, n=4-10/group). (D) Fecal water content is not altered by AM251 (1 mg/kg) or AM4113 (1 mg/kg) n=6-7/group. * p<0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Effect of the inverse agonist/antagonist AM251 and the neutral antagonist AM4113 on whole gut transit time. (A) shows the whole gut transit time when the antagonists are given 1 mg/kg (n=5/group), and (B) when the antagonists are given 5 mg/kg (n=8-9/group). * p<0.05; ** p < 0.01.

Figure 6

Neither AM251 (A) nor AM4113 (B) affected I_sc responses to electrical field stimulation or (C) serosal forskolin (FSK, 10 μM) or carbachol (CCh, 60 μM). (V – vehicle; n = 7 mice in A, n = 6 mice in B, n = 9-13 mice in C.