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. 2013 Aug;71(100):255–263. doi: 10.1016/j.neuropharm.2013.03.029

A role for O-1602 and G protein-coupled receptor GPR55 in the control of colonic motility in mice

Kun Li b,g,1, Jakub Fichna b,c,1, Rudolf Schicho b,d, Dieter Saur e, Mohammad Bashashati h, Ken Mackie f, Yongyu Li g, Andreas Zimmer h, Burkhard Göke a, Keith A Sharkey i, Martin Storr a,h,*
PMCID: PMC3677091  PMID: 23603203

Abstract

Objective

The G protein-coupled receptor 55 (GPR55) is a novel cannabinoid (CB) receptor, whose role in the gastrointestinal (GI) tract remains unknown. Here we studied the significance of GPR55 in the regulation of GI motility.

Design

GPR55 mRNA and protein expression were measured by RT-PCR and immunohistochemistry. The effects of the GPR55 agonist O-1602 and a selective antagonist cannabidiol (CBD) were studied in vitro and in vivo and compared to a non-selective cannabinoid receptor agonist WIN55,212-2. CB1/2−/− and GPR55−/− mice were employed to identify the receptors involved.

Results

GPR55 was localized on myenteric neurons in mouse and human colon. O-1602 concentration-dependently reduced evoked contractions in muscle strips from the colon (∼60%) and weakly (∼25%) from the ileum. These effects were reversed by CBD, but not by CB1 or CB2 receptor antagonists. I.p. and i.c.v. injections of O-1602 slowed whole gut transit and colonic bead expulsion; these effects were absent in GPR55−/− mice. WIN55,212-2 slowed whole gut transit effects, which were counteracted in the presence of a CB1 antagonist AM251. WIN55,212-2, but not O-1602 delayed gastric emptying and small intestinal transit. Locomotion, as a marker for central sedation, was reduced following WIN55,212-2, but not O-1602 treatment.

Conclusion

GPR55 is strongly expressed on myenteric neurons of the colon and it is selectively involved in the regulation of colonic motility. Since activation of GPR55 receptors is not associated with central sedation, the GPR55 receptor may serve as a future target for the treatment of colonic motility disorders.

Keywords: Cannabidiol, Cannabinoid, Colon, Gastrointestinal motility, GPR55

Abbreviations: CBD, cannabidiol; CB1, cannabinoid-1; CB2, cannabinoid-2; cDNA, complementary DNA; COX-2, cyclooxygenase-2; GE, gastric emptying; GPR55, G protein-coupled receptor 55; EFS, electrical field stimulation; ECS, endocannabinoid system; FAAH, fatty acid amide hydrolase; GI, gastrointestinal; i.c.v., intracerebroventricular administration; i.p., intraperitoneal; KRS, Krebs–Ringer solution; LMMP, longitudinal muscle-myenteric plexus layer; MAGL, monoacylglycerol lipase; PPARα, peroxisome proliferator-activated receptor-alpha; RT-PCR, reverse transcription polymerase chain reaction; TRPV1, transient receptor potential vanilloid 1

Highlights

  • •

    G protein-coupled receptor 55 (GPR55) is a binding site for cannabinoids.

  • •

    No conclusive information was available on function of GPR55 in the GI tract.

  • •

    We found that targeting GPR55 at peripheral or central sites slows GI motility.

  • •

    Slowing effect of GPR55 activation on GI motility is primarily observed in colon.

  • •

    Targeting GPR55 may be a future tool for treatment of colonic motility disorders.

1. Introduction

The prevalence of functional gastrointestinal disorders (FGID), such as irritable bowel syndrome (IBS), is currently estimated at 10–20%. It has a tendency to increase, in particular in the societies adopting Western style of living (Philpott et al., 2011). Symptoms manifested by FGID patients – predominantly altered motility patterns, stool inconsistency and bloating are not life-threatening, but are often associated with abdominal pain and have a negative impact on life quality. Thus, such disturbances have become a heavy economic burden due to increased work absenteeism, as well as increased use of health care services (Drossman, 2006). Current understanding of the pathogenesis of FGID and their clinical resolution are unsatisfactory. So far, hypotheses suggest low grade inflammation (Mayer and Collins, 2002; Philpott et al., 2011), food allergy (Atkinson et al., 2004) or disturbances in the bi-directional communication between the gut and the central nervous system (CNS) (Fichna and Storr, 2012). New therapeutic strategies, alleviating motility disturbances and pain without adverse, mainly related to the central nervous system side effects are therefore urgently needed.

The endocannabinoid system (ECS) consists of cannabinoid (CB)1 and CB2 receptors, their endogenous ligands anandamide and 2-arachidonylglycerol, and the synthesizing and degrading enzymes for these ligands. Cannabinoids and the ECS are involved in the regulation of GI motility in physiological and pathophysiological conditions (Izzo et al., 2001; Massa et al., 2005; Storr et al., 2008; Pertwee et al., 2010). In particular, the involvement of the cannabinoid receptors CB1 and CB2 and the endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) has been reported (Pinto et al., 2002; Duncan et al., 2008; Storr et al., 2009). In the last two decades, in vitro and in vivo studies have revealed roles for both central and peripheral CB receptors in the control of GI motility (Coutts and Izzo, 2004; Izzo et al., 2001; Storr and Sharkey, 2007). Interestingly, not all cannabinoid effects on GI motility can be explained by actions on CB1 and CB2 receptors and numerous additional sites of actions have been suggested. These include transient receptor potential vanilloid 1 (TRPV1) receptors, peroxisome proliferator-activated receptor-alpha (PPARα) and cyclooxygenase-2 (COX-2) inhibition (Pertwee et al., 2010; Piomelli, 2003).

Recently, an orphan G protein-coupled receptor 55 (GPR55) was shown to be a binding site for cannabinoids and became a likely candidate for mediating some of the previously unexplained non-CB1, non-CB2 effects induced by certain cannabinoids (Moriconi et al., 2010; Pertwee, 2007; Ross, 2009). Radioligand binding studies demonstrated GPR55 binding for both endocannabinoids and synthetic cannabinoids that may also be agonists or antagonists at CB1 or CB2 receptors, or both. The atypical synthetic cannabinoid O-1602 was found to activate GPR55 with negligible binding to CB1 and CB2 and is considered as a selective GPR55 agonist (Ryberg et al., 2007; Whyte et al., 2009; Johns et al., 2007; Schicho et al., 2010). Recent data from a transgenic GPR55−/− mouse model further supports O-1602 as a GPR55 agonist (Whyte et al., 2009). Little information is available on antagonists at the GPR55 receptor, although cannabidiol (CBD) appears to be a selective GPR55 receptor antagonist (Pertwee, 2007; Ryberg et al., 2007; Whyte et al., 2009; Thomas et al., 2007).

Localization of GPR55 is rarely studied. However, convincing data shows that GPR55 mRNA is expressed in the brain (Sawzdargo et al., 1999). The GPR55 activation was reported to have effects on osteoclast function, bone density and cancer cell proliferation, but surprisingly there is no conclusive information yet available shedding light on the localization and function of GPR55 in the gastrointestinal (GI) tract (Whyte et al., 2009; Sawzdargo et al., 1999; Pineiro et al., 2010; Andradas et al., 2010; Lin et al., 2011).

The present study aimed at identifying whether GPR55 is expressed in the GI tract and where it is localized. We also investigated the role of the GPR55 receptor in the regulation of mouse GI motility in vitro and in vivo, utilizing a selective GPR55 agonist, O-1602 and the antagonist, CBD. The effects were further analysed employing a well-characterized cannabinoid receptor agonist WIN55,212-2 and selective CB1 and CB2 antagonists. The involvement of respective receptors in the action of O-1602 and CBD was also studied in the CB1/2−/− and GPR55−/− mice. Finally, the action of GRP55-selective compounds in the central nervous effects was characterized.

2. Material and methods

2.1. Animals

Male CD1, GPR55−/− and CB1/2−/− mice and their littermates on a C57Bl/6 background were used throughout the study (22–26 g). The CD1 mice were purchased from Charles River (Sherbrooke, QC, Canada). The GPR55−/− mice were acquired from the Texas Institute of Genomic Medicine (TIGM, Houston, TX) and bred at the animal facilities of the Department of Psychological and Brain Sciences, Indiana University, Bloomington, USA (Wu et al., 2010). The CB1/2−/− mice were bred at the mouse facility at the University of Calgary, Canada from a pair of animals provided by A. Zimmer (University of Bonn, Germany). CD1 mice for the PCR experiments were housed in the animal facility of the Technical University of Munich, Germany.

All experiments were performed in CD1 mice unless otherwise stated. Animals were matched by age and body weight. Mice were housed at a 12:12-h light–dark cycle in sawdust coated plastic cages with access to standard laboratory chow and tap water ad libitum. Mice were allowed 1 week of acclimatization prior to use. For in vitro experiments mice were sacrificed by cervical dislocation and their GI tract was isolated and washed with normal solution. All experiments were approved by the University of Calgary Animal Care Committee and the experiments were performed in accordance with institutional animal ethics committee guidelines following the guidelines established by the Canadian Council of Animal Care. The GPR55−/− mouse experiments were approved by the Indiana University (Bloomington, IN, USA) Institutional Animal Care and Use Committee.

All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available.

2.2. RNA isolation and RT-PCR

To determine GPR55 mRNA expression in mouse GI tract, total RNA was extracted from the longitudinal muscle-myenteric plexus (LMMP) and the mucosal layer of the mouse colon and ileum. Tissue preparation: Adult CD1 mice were killed by cervical dislocation. The small and large intestine was removed and cleaned in ice-cold PBS. Attached mesenterial fat was removed and the longitudinal muscle layer with attached myentric plexus (LMMP) was removed from the circular muscle layer by peeling. Subsequently, the mucosa was removed from the circular muscle layer by scraping. Tissues were cut into small pieces, immediately frozen in liquid nitrogen and stored at −80 °C until use. RNA isolation, reverse transcription, PCR amplification, and agarose gel electrophoresis are described in detail in the Appendix.

2.3. Immunohistochemistry

Whole mount preparations of the ileum and the distal colon were obtained for the immunohistochemical detection of GPR55 using standard approaches. The procedures were as follows: after incubating in a solution of 0.1 M PBS, 4% donkey serum, and 0.1% Triton-X-100 for 1.5 h, the whole mounts were exposed to rabbit anti-GPR55 (K. Mackie), 1:800, overnight at 4 °C. To visualize immunoreactivity, a fluorophore-conjugated secondary antibody (anti-rabbit Cy3; 1:800; Jackson ImmunoResearch, West Grove, PA, USA) was used. The specimens were examined under a Zeiss Axioplan brightfield/fluorescence microscope and photographed with a digital camera (Sensys, Photometrics, Tucson, AZ, USA). Brightness and contrast of the images were adjusted using Adobe Photoshop®. Negative controls in which the primary antibody was omitted and specificity controls with antigen–antibody pre-absorption were performed.

Paraffin-embedded sections of human colon were obtained from the tissue bank of the University of Graz, Austria. Sections were deparaffinized, microwaved for 2 × 5 min cycles in 10 mM citrate buffer and then processed by the ABC method (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's protocol. Sections were incubated with rabbit anti-GPR55 (1:800; Cayman Chemical Company, Ann Arbor, Michigan, USA), visualized with 3-3′-diaminobenzidine and counterstained with haematoxylin. The specificity of the antibody was tested by omitting the primary antibody and by incubating the GPR55 antibody with blocking peptide provided by the manufacturer (Cayman Chemical Company, Ann Arbor, Michigan, USA).

2.4. Isolated intestinal segments

The experiments on isolated intestinal segments were performed using a setup described previously. At the beginning of each experiment, 0.5 g tension was applied, the tissue was incubated for 30 min under standard conditions and then stimulated with bethanechol (10−5 M) in order to obtain a maximal contraction (100% contraction). The tissue was then washed 3 times and the experiment was started. In a first set of experiments, tissues were exposed to either the GPR55 agonist O-1602 (10−10–10−6 M), the cannabinoid receptor agonist WIN55,212-2 (10−10–10−6 M), the CB1 antagonists AM251 and SR141716A (both 10−7 M), the CB2 antagonist AM630 (10−7 M), and the GPR55 antagonist CBD (10−7 M). Changes in tension or basal activity were then recorded for 60 min.

Trains of electrical field stimulation (EFS; train duration 10 s, 8 Hz, 0.5 ms pulse duration, 40 V) were applied every 2 min. Following 60 min of stimulation, drugs were applied in a cumulative manner to the organ bath in 20 min intervals. The following drugs were applied: O-1602 (10−10–10−6 M) and WIN55,212-2 (10−10–10−6 M). In separate experiments, WIN55,212 and O-1602 were tested in a cumulative manner as detailed above and an antagonist was given 30 min prior. The following antagonists were tested: AM251 (10−7 M), SR141716A (10−7 M), AM630 (10−7 M), and CBD (10−8 M). In additional experiments the effects of WIN55,212-2 and O-1602 were characterized in tissue sections obtained from CB1/2−/− mice. The effects of WIN55,212-2 and O-1602 were also investigated under non-adrenergic, non-cholinergic conditions, in the presence of atropine and guanethidine (both 10−6 M) (Mule et al., 2007b, 2007a).

2.5. Whole gut transit time

Whole gut transit was measured as reported previously (Storr et al., 2010). O-1602 (5 or 10 mg/kg), WIN55,212-2 (1 mg/kg), or vehicle were injected intraperitoneally (i.p.) 20 min prior to Evans blue administration. In subsequent experiments, mice received AM251 (0.1 mg/kg) or CBD (0.5 mg/kg) i.p. 15 min prior to O-1602 (5 or 10 mg/kg), WIN55,212-2 (1 mg/kg) or vehicle. Doses of antagonists were chosen based on the literature and preliminary experiments (Storr et al., 2010).

To investigate possible central effects of O-1602 and WIN55,212-2 were given in a dose of 10 μg/kg by intracerebroventricular (i.c.v.) administration (Haley and McCormick, 1957) 5 min before the gavage of Evans blue suspension.

To investigate the involvement of GPR55 receptors, GPR55−/− mice and wild type littermates were injected i.p. with O-1602 (10 mg/kg), WIN55,212-2 (1 mg/kg) or vehicle and whole gut transit time was measured as detailed above.

2.6. Gastric emptying and small intestinal transit time (geometric centre)

Gastric emptying and geometric centre were performed according to techniques described earlier (Fichna et al., 2009). O-1602 (10 mg/kg), WIN55,212-2 (1 mg/kg) or vehicle were injected i.p. 20 min before the administration of the marker solution.

2.7. Colonic expulsion

Distal colonic expulsion was measured as previously described (Yuce et al., 2007). O-1602 (10 mg/kg), WIN55,212-2 (0.3 mg/kg), or vehicle were injected i.p. 20 min prior to bead insertion. In separate experiments AM251 (0.1 mg/kg) or CBD (0.5 mg/kg) were injected i.p 15 min prior to O-1602 or WIN55,212-2.

To investigate the involvement of GPR55 receptors, GPR55−/− mice and wild type littermates were injected i.p. with O-1602 (10 mg/kg), WIN55,212-2 (1 mg/kg) or vehicle and colon bead expulsion time was measured as detailed above.

2.8. Locomotor activity

Ambulatory locomotor activity was measured 30 min after O-1602 (10 mg/kg, i.p.) and WIN55,212-2 (1 mg/kg, i.p.) or 5 min after O-1602 (10 μg/kg, i.c.v.) and WIN55,212-2 (10 μg/kg, i.c.v.) using an infrared beam activity monitor (Columbus Instruments, Columbus, OH, USA). Mice were pre-exposed to the recording equipment in the morning and the experiments were performed in the afternoon of the same day. Each individual mouse was placed in the apparatus and the ambulatory count was recorded over a 10 min period. Movement of the mice was recorded as the ambulatory activity count when the infrared beams were sequentially broken.

2.9. Drugs

WIN55,212-2, O-1602, CBD, AM251, AM630 and SR141716A were purchased from Tocris Bioscience (Burlington, ON, Canada). The compounds were dissolved in ethanol and further diluted in sterile saline with Tween 80 (2%) and DMSO (4%). Atropine, guanethidine, Evans blue and Gum Arabic were purchased from Sigma–Aldrich (Oakville, ON, Canada). In separate experiments the vehicles used were tested in the in vitro and the in vivo tests and did not show significant effects on the observed parameter.

2.10. Statistics

Each set of in vitro experiments was performed on a minimum of 6 independent preparations from a minimum of 3 different animals and the data are expressed as percent change compared to respective vehicle control. All results are expressed as mean ± SEM of experiments in n = 6–10 mice. Comparisons between two sets of data were made by Student's t-test for unpaired data or one-way ANOVA followed by the Bonferroni post hoc test for multiple treatments. P values < 0.05 were considered significant.

3. Results

3.1. Expression of GPR55 mRNA in ileum and colon

Using RT-PCR, GPR55 mRNA expression was found in the LMMP and the mucosa of the ileum and colon (Fig. 1A). Quantitative analysis showed that there was a relatively low expression of GPR55 mRNA in the LMMP of the ileum (Fig. 1B).

Fig. 1.

Fig. 1

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GPR55 expression as determined by RT-PCR in mouse ileum and colon. (A) Bands of GPR55 mRNA expression in ileum LMMP (1), ileum mucosa (2), colon LMMP (3), colon mucosa (4) and negative control (5). (B) Quantitative analysis of bands indicating relative expression of GPR55 mRNA normalized to GAPDH mRNA. GPR55 immunofluorescence was hardly detected in the myenteric plexus of mouse ileum (C), but was clearly present in the colon (D). In the mouse colon positive signalling of GPR55 was visible in both, nerve fibres and ganglion cells. Calibration bar = 50 μm. GPR55 immunoreactivity was also detected in the myenteric plexus of human colon sections (E; arrows) and in sections of the mouse colon (G; arrows). Pre-absorption controls with blocking peptide are shown for human (F) and mouse (H) myenteric plexus to prove the specificity of the antibody. Calibration bar: 50 μm; cm (circular muscle), lm (longitudinal muscle).

3.2. GPR55 expression in the myenteric plexus of the ileum and colon

The distribution of GPR55 immunoreactivity in the myenteric plexus of mouse ileum and colon is shown in Fig. 1C and D. GPR55 immunoreactivity was found on myenteric neurons of the colon and on nerve fibres and the ganglion cell bodies (Fig. 1D). In contrast, in the myenteric plexus of mouse ileum, GPR55 immunoreactivity was rather low (Fig. 1C).

GPR55 immunoreactivity was also detected in the myenteric plexus of human colon sections obtained from healthy controls (Fig. 1G, H).

3.3. The effects of the GPR55 agonist O-1602 on ileal and colonic contractility in vitro

None of the used drugs had effects on basal tension or basal activity of the ileal or colonic preparations in vitro (data not shown).

O-1602 and WIN55,212-2 reduced EFS evoked contractile responses in ileal and colonic segments in a concentration-dependent manner (Fig. 2), but the effects in the ileum were observed only at the highest concentration used. The maximal inhibitory effect of O-1602 (10−6 M) was ∼ −25% in the ileum and ∼ −60% in the colon (Fig. 2B), whereas the maximal effect observed for WIN55,212-2 (10−6 M) was ∼40% for ileum and colon (Fig. 2C).

Fig. 2.

Fig. 2

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Inhibitory effect of O-1602 and WIN55,212-2 on EFS-induced contractions in vitro. A) Representative tracings for mouse ileum and colon. B) Effects of O-1602 alone and after pre-incubation with AM251 (10−7 M) or AM630 (10−7 M) in mouse ileum and colon. C) Effects of WIN55,212-2 alone and with AM251 (10−7 M) or AM630 (10−7 M) pre-incubation in mouse ileum and colon. D) Effects of O-1602 alone and with AM251 (10−7 M) or SR141716A (10−7 M) pre-incubation in ileum and colon of CB1,2−/− mice. Data show mean ± SEM for n = 6–11. *P < 0.05 for drug vs. vehicle treatment; #P < 0.05 for antagonist + drug vs. drug treatment.

The inhibitory effect of O-1602 was not changed in the presence of either AM251 or AM630 (both 10−7 M), suggesting that CB1 and CB2 receptors are not involved in the actions of O-1602 (Fig. 2B). AM251 and AM630 (both 10−7 M) had no significant effect on ileum or colon contractility (AM251 10−7 M: ileum: 92.9 ± 3.3; colon: 116.7 ± 12.6; AM630 10−7 M: ileum: 100.7 ± 2.1; colon: 94.9 ± 5.8; n = 6–7). In contrast, the effect of WIN55,212-2 was blocked in presence of AM251 (10−7 M), but not by AM630 (10−7 M) in both ileum and colon (Fig. 2C), showing that the CB1 receptor mediates the actions of WIN55,212-2, as previously demonstrated (Storr et al., 2010).

The effects of O-1602 on ileum and colon were not altered in CB1/2−/− mice. AM251 and SR141716A (both 10−7 M) had no significant effect on ileum or colon contractility. In addition, AM251 and SR141716A (both 10−7 M), did not alter the effects of O-1602 in CB1/2−/− mice (Fig. 2D).

Furthermore, we tested the effect of O-1602 on EFS induced contractions in the colon under NANC conditions. Under these conditions the effect of O-1602 on EFS induced colonic contractions was unchanged (data not shown).

Vehicle or antagonists alone at the concentrations used had no effect on the EFS evoked contractile responses in ileal or colonic segments.

3.4. CBD antagonizes the inhibitory effect of O-1602 in vitro

The GPR55 antagonist CBD (10−9 and 10−8 M) had no effect on basal tension or basal activity of mouse ileum or colon. In the colon, CDB reduced EFS induced contractions at 10−7 M (−37.6 ± 5.7%; P < 0.05; n = 6), whereas no such effect was seen in the ileum (−8.9 ± 5.1%; n.s.; n = 6). No significant changes in the EFS responses were seen at 10−9 M and 10−8 M, therefore these concentrations were used against O-1602.

CBD (10−9 and 10−8 M) blocked the inhibitory effect of O-1602 on EFS induced contractions in mouse ileum (Fig. 3A) and CBD at 10−8 M, but not at 10−9 M blocked the inhibitory effects of O-1602 in mouse colon (Fig. 3B). CBD at 10−8 M did not block the inhibitory effect of WIN55,212-2 on EFS induced contractions in neither mouse ileum or colon (Fig. 3C and D).

Fig. 3.

Fig. 3

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Effects of O-1602 alone or with CBD (10−8 and 10−9 M) pre-incubation in mouse ileum (A) and colon (B). Effects of WIN55,212-2 alone and with CBD (10−8 M) pre-incubation in mouse ileum (C) and colon (D). Data show mean ± SEM for n = 6–12. *P < 0.05 for drug vs. vehicle treatment; #P < 0.05 for antagonist + drug vs. drug treatment.

3.5. Influence of O-1602 and WIN55,212,2 on whole gut transit in vivo

Both, O-1602 (10 mg/kg) and WIN55,212-2 (1 mg/kg) slowed whole gut transit following an i.p. injection (Fig. 4A). Whereas AM251 (0.1 mg/kg i.p.) did not change the effect of O-1602, it significantly reversed the effect of WIN55,212-2. CBD (0.5 mg/kg i.p.) significantly reversed the effect of O-1602 on whole gut transit, whereas it had no effect on the actions of WIN55,212-2 (Fig. 4A). Neither AM251, nor CBD alone altered the whole gut transit time at the doses tested (data not shown).

Fig. 4.

Fig. 4

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O-1602 (10 mg/kg i.p.; 10 μg i.c.v.) and WIN55,212-2 (1 mg/kg i.p.; 10 μg i.c.v.) given i.p. (A) or i.c.v. (B) slowed whole gut transit. A: CBD (0.5 mg/kg) blocked the effects of O-1602, but not the effects of WIN55,212. AM251 (0.1 mg/kg) blocked the effects of WIN55,212, but not the effects of O-1602 i.p. B: CBD (50 μg i.c.v.) blocked the effects of O-1602 and WIN55,212. AM251 (20 μg i.c.v.) blocked the effects of WIN55,212, but not the effects of O-1602 i.p. C: O-1602 (10 mg/kg) and WIN55,212-2 (0.3 mg/kg) given i.p. slowed whole gut transit. CBD (0.5 mg/kg) blocked the effects of O-1602, but not the effects of WIN55,212. AM251 (0.1 mg/kg) blocked the effects of WIN55,212, but not the effects of O-1602 i.p. Data show mean ± SEM for n = 6–12. *P < 0.05 for drug vs. vehicle treatment; #P < 0.05 for antagonist + drug vs. drug treatment.

When given i.c.v., O-1602 and WIN55,212-2 (both 10 μg/kg) inhibited whole gut transit (Fig. 4B). AM251 (50 μg/kg, i.c.v.) significantly reversed the inhibitory effect of WIN55,212-2, but not O-1602. CBD (20 μg/kg, i.c.v.) significantly reversed the inhibitory effect of O-1602 on whole gut transit. Interestingly, CBD (20 μg/kg, i.c.v.) also significantly reversed the inhibitory effect of WIN55,212-2 on whole gut transit (Fig. 4B). Neither AM251, nor CBD alone altered whole gut transit time after i.c.v. administration (data not shown).

3.6. Influence of O-1602 and WIN55,212,2 on gastric emptying and small intestinal transit time in vivo

WIN55,212-2 (1 mg/kg, i.p.) delayed gastric emptying by 37.5 ± 5.8% (P < 0.05; n = 8), whereas O-1602 (10 mg/kg, i.p.) had no significant effect (+11.0 ± 3.2%; n.s.; n = 7).

WIN55,212-2 (1 mg/kg, i.p.) inhibited small intestinal transit time by 25.4 ± 3.9% (P < 0.05; n = 8), whereas O-1602 (10 mg/kg, i.p.) did not produce any significant effect (−7.2 ± 5.1%; n.s.; n = 7).

3.7. Effect of O-1602 and WIN55,212,2 on colonic propulsion in vivo

O-1602 (10 mg/kg) and WIN55,212-2 (0.3 mg/kg) administered i.p. slowed colonic bead expulsion in mice (Fig. 4C). AM251 (0.1 mg/kg) did not influence the effect of O-1602 (+11.2 ± 6.9%; n.s.; n = 7), but blocked the inhibitory effect of WIN55,212-2 on colonic bead expulsion time (Fig. 4C). CBD (0.5 mg/kg) blocked the inhibitory effect of O-1602, but not of WIN55,212-2 on colonic bead expulsion (Fig. 4C).

Neither AM251, nor CBD alone altered the colonic propulsion in vivo at the doses tested (data not shown).

3.8. Effect of O-1602 and WIN55,212,2 in GPR55−/− mice in vivo

Both, O-1602 (10 mg/kg) and WIN55,212-2 (1 mg/kg) slowed whole gut transit following an i.p. injection in GPR55+/+ mice (Fig. 5A). Whole gut transit time in GPR55+/+ mice did not differ compared to GPR55−/− mice (Fig. 5A). The effect of O-1602 on whole gut transit time was absent in GPR55−/− mice, whereas the effect of WIN55,212-2 was maintained (Fig. 5A).

Fig. 5.

Fig. 5

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O-1602 (10 mg/kg) and WIN55,212-2 (1 mg/kg) given i.p. slowed whole gut transit in C57/Bl6 mice (A). WIN55,212-2 (1 mg/kg), but not O-1602 (10 mg/kg) slowed whole gut transit in GPR55−/− mice (A). O-1602 (10 mg/kg) and WIN55,212-2 (1 mg/kg) given i.p. slowed colonic bead expulsion time in C57/Bl6 mice (A). WIN55,212-2 (1 mg/kg), but not O-1602 (10 mg/kg) slowed colonic bead expulsion time in GPR55−/− mice (A). Data show mean ± SEM for n = 6–8. *P < 0.05 for drug vs. vehicle treatment. C57/Bl6 (wildtype) and GPR55−/− were matched by age and body weight.

Similarly, O-1602 (10 mg/kg) and WIN55,212-2 (1 mg/kg) administered i.p. slowed colonic bead expulsion in GPR55+/+ mice (Fig.5B). Colonic bead expulsion time in GPR55+/+ mice did not differ compared to GPR55−/− mice (Fig. 5B). The effect of O-1602 on colonic bead expulsion time was absent in GPR55−/− mice, whereas the effects WIN55,212-2 were unaffected (Fig. 5B).

3.9. Effect of O-1602 and WIN55,212-2 on locomotor activity

Ambulatory locomotor activity was measured 30 min after O-1602 (10 mg/kg) and WIN55,212-2 (1 mg/kg) injected i.p. or 5 min after O-1602 (10 μg) and WIN55,212-2 (10 μg) injected i.c.v., respectively. O-1602 had no effect on the movements of the mice, in contrast WIN55,212-2 significantly decreased the locomotor activity (Fig. 6).

Fig. 6.

Fig. 6

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Effects of O-1602 and WIN55,212-2 given i.p. or i.c.v. on mouse locomotor activity. O-1602 was given 10 mg/kg i.p. or 10 μg i.c.v. and WIN55,212-2 was given 1 mg/kg i.p. or 10 μg i.c.v. and locomotion was monitored for 30 min. Data represent as mean ± SEM for n = 6–8. *P < 0.05 compared to vehicle only treatment.

4. Discussion

The ECS is involved in the regulation of GI motility under physiological and pathophysiological conditions. The discovery of new elements of the ECS is of particular interest, since it sets the stage for the development of the therapeutic potential of the system (Schicho and Storr, 2010). However, the usefulness of targeting the ECS is presently limited by psychotropic side effects of CB1 receptor agonists or antagonists (Schicho and Storr, 2011). The recent discovery that some cannabinoids exert strong binding at the GPR55 receptor and the observation that GPR55 exhibits a number of key differences in comparison with classical CB receptors has sparked considerable interest in this novel cannabinoid binding site (Johns et al., 2007; Sharir and Abood, 2010). In the present study we have identified a previously unknown distribution and function of GPR55 in the GI tract of mice and humans, which may be crucial for the future use of GPR55 agonists in the clinical treatment of IBS and other FGIDs associated with GI hypermotility.

Using RT-PCR we demonstrated the expression of GPR55 receptor mRNA in the ileum and colon of mice, which is in good agreement with previous reports (Lin et al., 2011). The quantitative analysis showed the abundance of GPR55 mRNA in the mucosa of the ileum and colon. In contrast, in LMMP preparations the GPR55 expression was significantly higher in the colon compared to the ileum, where expression is relatively low. Using immunohistochemistry we further revealed that GPR55 immunoreactivity can be localized on ganglion cells and nerve fibres in the myenteric plexus of the colon, but hardly in the ileum myenteric plexus, strengthening our findings based on the RT PCR technique.

We were also interested whether the GPR55 localized on myenteric neurons is involved in the regulation of intestinal motility and whether the differences in receptor expression translates to differences in pharmacological response to GPR55 activation. For the studies on the in vitro and in vivo motility we used O-1602, a GPR55 agonist, and a GPR55-selective antagonist CBD (Ryberg et al., 2007). We found no effect of O-1602 or CBD on basal tone or on pharmacologically stimulated smooth muscle, suggesting that a direct action of O-1602 on the smooth muscle was unlikely. These findings were supported by our immunohistochemical studies showing the lack of GPR55 staining on smooth muscle cells.

O-1602 significantly reduced electrically evoked contractions suggesting a neural site of the GPR55 receptor. This effect was far more pronounced in the colon than in the ileum, showing distinct regional differences of action of O-1602 in the GI tract. In the colon the effect of O-1602 was comparable to that of classical cannabinoids, such as the CB1/CB2 agonist WIN55,212-2. Interestingly, the difference between the effects observed in the ileum and the colon was less pronounced for WIN55,212-2 than O-1602, suggesting significant differences between the mechanisms by which these different classes of cannabinoids exert their action.

For atypical cannabinoids, such as O-1602, the responding receptors are only poorly characterized. Functional studies indicate that atypical cannabinoids interact with CB1, CB2 and GPR55 receptors, as well as other structures. In our hands, the actions of O-1602 were neither altered in the presence of a CB1 or a CB2 antagonist, nor observed in tissues of CB1,2−/− mice. This indicates that the O-1602-mediated effects are independent of the classical cannabinoid receptors. As some recent studies suggested that commonly used CB1 antagonists may bind and have effects at receptors other than CB1, we were also interested whether these antagonists would influence O-1602 effects in the GI tissue of CB1,2−/− mice. In our hands such effects were not observed. Our pharmacological in-vitro studies confirm a recent publication where comparable experiments resulted in comparable effects and where O-1602 effects were additionally shown to be absent in tissues from GPR55−/− mice (Ross et al., 2012). The logical next question is whether these GPR55 mediated effects are of relevance in in vivo motility and to address this, we performed in-vivo motility studies in wild type and in GPR55−/− mice.

CBD was recently reported to have antagonist effect at the GPR55 receptor (Ryberg et al., 2007). In our study the in vitro effects of O-1602 in the ileum and colon were antagonized by CBD, confirming that GPR55 mediates its actions. CBD did not alter the effects of WIN55,212-2 indicating that the WIN55,212-2 effect on GI motility does not involve GPR55 activation, which is in agreement with receptor binding studies suggesting that WIN55,212-2 does not bind to the GPR55 receptor (Ryberg et al., 2007).

We further investigated the actions of O-1602 in vivo. O-1602 slowed whole gut transit and delayed colonic expulsion and, in contrast to the effects of WIN55,212-2 on in vivo motility, these effects were not sensitive to CB1 receptor antagonists. We also extended our in vitro observations to the in vivo finding that the effects of O-1602, but not those of WIN55,212, were antagonized by CBD. Again, this supports the idea of GPR55 mediating the inhibitory effects of O-1602 on GI motility.

Throughout our studies we used CBD under the assumption that it binds to the GPR55 receptor and has antagonist properties at the GPR55 receptor, as previously suggested (Whyte et al., 2009). To clarify whether the GPR55 receptor is involved in the O-1602-mediated effects on GI motility, we performed whole gut transit time and colonic expulsion experiments in GPR55−/− mice. Whereas the WIN55,212-2 effect was unchanged in GPR55−/− mice, O-1602 was not effective in GPR55−/− mice proving that O-1602 slows GI motility via GPR55 receptors. This important novel observation suggests that the GPR55 receptor is a promising future target for the treatment of GI motility disorders, especially when the goal is slowing of GI motility and transit. These experiments in GPR55−/− mice are of special importance since due to the lack of highly specific or highly selective GPR55 receptor agonists and antagonists, pharmacological studies on GPR55 functioning has to be carefully discussed. In a recent study where we investigated GPR55 involvement in postoperative ileus, we were able to prove that GPR55 expression is higher in the inflammatory state, but we were not able to characterize GPR55 effects on neurotransmission since in this post-inflammatory state the GRR55 agonist and antagonist had same directed effects, which limited any further interpretation (Lin et al., 2011). CBD was identified to reduce inflammatory hypermotility in mice (Capasso et al., 2010) which was again seen in the study where postoperative ileus was studied. CBD effects were at that time not mediated by CB1 or CB2 involving pathways and it is furthermore unlikely that GPR55 was the mediating receptor since the CBD effect was shown to be located on smooth muscle cells and our present study shows that GPR55 is not located on smooth muscle cells (Capasso et al., 2010).

In contrast to other cannabinoid drugs, GPR55 activation may not be limited by central sedating side effects. This is highlighted by our observation that WIN55,212-2, but not O-1602 reduced ambulatory locomotion regardless whether it was given peripherally or centrally.

One final result warrants further attention. O-1602 slowed GI motility by acting both at peripheral and central sites, similarly to CB1 receptor agonists (Izzo et al., 2000). Interestingly, we observed that the effects of WIN55,212-2, when given directly into the CNS, can be reversed by CBD, suggesting that GPR55 receptors are involved in the central slowing effects of cannabinoids. Since WIN55,212-2 does not bind to GPR55 receptors (Johns et al., 2007), this effect may be due to downstream activation of GPR55 receptors, following central activation of CB1 binding sites, but it needs to be addressed further in a separate study. However, the pertinent finding in this context here deserves further attention since it suggests future cannabinoid receptor targeting approaches devoid of the undesired CNS side effects, commonly associated with the activation of CB1 receptor.

In summary, our observations suggest that targeting the GPR55 receptor may be a promising future tool for the treatment of FGID patients. We have shown that the activation of GPR55 produced potent antimotility effects similar to those of the cannabinoid receptor agonists. However, they are clearly lacking the adverse side effects related to the activation of the receptors in the CNS. These observations add to the potentially beneficial role of GPR55 agonists in GI inflammation and endocrine pathophysiology and set a new pharmacological approach in the clinical treatment of motility disturbances.

Competing interests

No author declares a conflict of interest.

KL, JF, MB, YYL, RS, KAS, MS: the conception and design of the study; KL, JF, DS, MB, BG, RS, MS: acquisition, analysis and interpretation of data; JF, KAS, MS: drafting the manuscript; KL, JF, DS, MB, RS, YYL, KAS, BG, MS: critical revision of the manuscript for important intellectual content; JF, MS: final approval of the version to be submitted.

Funding

Supported by the University of Calgary Research Grant Committee (to MS), the Deutsche Forschungsgemeinschaft (DFG; STO 645-6-1 to MS) and the Canadian Institutes of Health Research (KAS). KAS is an Alberta Heritage Foundation for Medical Research Medical Scientist and the Crohn's and Colitis Foundation of Canada Chair in Inflammatory Bowel Disease Research. JF is supported by the Iuventus Plus Program of the Polish Ministry of Science and Higher Education (#0119/IP1/2011/71). RS is supported by the Austrian Science Fund (FWF; P-22771).

Acknowledgements

We thank Ms. Winnie Ho from the University of Calgary, Canada for performing the genotyping of the CB1/2 receptor gene deficient mouse colony.

Appendix A. Supplementary data

The following is the supplementary data related to this article:

mmc1.doc (37.5KB, doc)

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