CANNABINOID 1 RECEPTOR SIGNALING IN COLONIC NOCICPETION IS ALTERED BY CHANGES OF THE ENDOCANNABINOID SYSTEM DURING COLITIS

Background: Abdominal pain is a debilitating symptom in patients with inflammatory bowel disease. Previously we have shown that cannabinoid 1 receptor (CB1R) agonists are less effective at inhibiting abdominal pain in mice with acute colitis compared to healthy mice. However, the mechanism behind this change is currently unknown.

Aims: To determine the role of endocannabinoids in the modulation of the ability of a CB1R agonist to inhibit colonic pain signaling during acute colitis.

Methods: Dextran sulfate sodium (DSS) induced colitis in male and female C57BL/6 mice. Visceromotor response (VMR) to colorectal distension (CRD) (20-80 µL) was measured using electromyography. Mice were injected intraperitoneally with vehicle or ACEA, a selective CB1R agonist, 30 min prior to CRD. Extracellular afferent nerve recordings in colonic flat sheet preparations were used to assess mechanosensitivity before and after superfusion of ACEA. Endocannabinoid concentrations (2-AG, AEA) of colon and serum were quantified using liquid chromatography mass spectrometry. Data were analyzed using a one or two-way ANOVA with Bonferroni test. N= number of mice; n=number of single axons.

Results: While ACEA (3 mg/kg) was previously found to inhibit VMR in healthy mice, but had no effect during acute colitis, an increased dose (10 mg/kg) significantly inhibited VMR to CRD in mice with colitis (-40.4% @ 80 µL, p<0.001, N=6) . Similarly, in afferent nerve recordings, ACEA (1 µM), previously inhibitory in healthy mice, had no effect during colitis (p=0.27, n=11, N=7). However, a higher concentration of ACEA (10 µM) significantly inhibited colonic mechanosensitivity (15.2 vs. 11.6 Hz; p<0.05, n=11, N=6). To explore why the CB1R agonist is less effective during acute colitis, mice were treated with AM-251, a CB1R antagonist (10 mg/kg, i.p.), once daily coinciding with the start of DSS administration until one day before sacrifice. Following AM-251 treatment during colitis, ACEA (1 µM) significantly reduced colonic mechanosensitivity (16.1 vs. 12.1 Hz; p<0.05, N=5, n=9). This 25% reduction in mechanosensitivity was similar to the 35% reduction induced by ACEA (1 µM) in healthy control mice (17.1 vs. 11.2 Hz; p<0.05, N=5, n=8) treated with the same regimen of AM-251. In healthy mice, 2-AG concentration in colonic tissue had a range of 4.42-6.91 ng/mL (N=4). While 2 DSS colitis mice had 2-AG levels similar to control mice, 4 DSS colitis mice had 2-AG levels of 10.27-193.57 ng/mL. Colonic tissue levels of AEA and serum endocannabinoids were similar in control and colitis mice.

Conclusions: CB1R agonists have reduced effectiveness on pain signalling during colitis. This may be due to release of endocannabinoids acting on the CB1R during acute colitis, leading to receptor desensitization, such that higher concentrations of exogenous CB1R are needed to inhibit colonic pain signalling.