Evidence for the putative cannabinoid receptor, GPR55, mediated inhibitory effects on intestinal contractility in mice

Abstract

Introduction

The biologically active constituents of Cannabis sativa (marijuana), cannabinoids, have been used or abused for decades for their psychoactive properties. Among over 70 distinct cannabinoid substances in marijuana, Δ⁹-tetrahydrocannabinol (THC),the main psychoactive ingredient[1], and cannabidiol are the most prevalent and best investigated[2]. Two seven-transmembrane G-protein coupled receptors: type 1 (CB1) cannabinoid receptors mediate most of the psychoactive effects and type 2 (CB2) receptors mediate immunological effects of these phytocannabinoids. Since the studies of Wagner and coworkers on endothelial anandamide receptor-mediated mesenteric vasodilation[3], it has been postulated that a non-CB1/ non-CB2 cannabinoid receptor or “abnormal cannabidiol (ABN-CBD)” receptor exists[4] and that this receptor could mediate effects of phytocannabinoids and endocannabinoids. Following the identification and cloning of novel human G-protein-coupled receptor 55 (GPR55)[5], several cannabinoid ligands were shown to bind to GPR55[6], suggesting that it could be a novel cannabinoid receptor. Based on [³⁵S]GTP_γS assays performed in transfected hGPR55-HEK293 cells, ABN-CBD and its analog O-1602, in which the pentyl side chain was shortened to a methyl group[4], were reported as GPR55 agonists[7–9]. In addition, JWH015 (1-propyl-2-methyl-3-( -naphthoyl)indole) is also considered to be a CB2-/GPR55 – receptor agonist[10] These compounds represent potentially useful pharmacological tools to investigate GPR55 receptors in different physiological systems.

The effects of cannabinoid receptor activation and the role of endocannabinoids in the gastrointestinal tract are now being realized as fairly extensive. CB1 receptor activation reduces intestinal motility, alleviates pain, and affects transient lower esophageal sphincter relaxations and emesis [11, 12]. Although the functional role of CB1 receptors has been extensively studied in the gastrointestinal tract, much less is known about the effects of the recently identified GPR55. Utilizing northern blot analysis or quantitative polymerase chain reaction methods that detect the receptor at the message level, high levels of human GPR55 mRNA transcripts have been found in brain regions and in several peripheral tissues, such as the ileum[5, 13]. Rat and mouse GPR55 homologs have also been detected in peripheral tissues including jejunum, ileum, and colon [8, 13, 14]. Recently Lin and co-workers have reported that GPR55 ligands could normalize lipopolysaccharide –induced motility disturbances in the gut [15]. However, the mechanisms by which gut motility is influenced by GPR55 receptors have yet to be verified in functional assays. Since cannabinoids are known to inhibit neurogenic contractions in the gut, we studied the effect of the reported GPR55 agonists, O-1602, an atypical cannabinoid, and JWH015 on the neurogenic (electrical field stimulated) contractions in mouse colon strips and also intended to determine the mechanisms of action of O-1602. In the present study, we used pharmacological and genetic manipulation approaches to test the hypothesis that activation of GPR55 receptors will result in inhibition of the neurogenic contractions in the mouse intestine.

Materials and Methods

Results

Inhibitory effect of the atypical cannabinoid O-1602 on neurogenic contractions in mouse colon

In spite of the fact that the gut expresses GPR55 receptors [13, 16], there has been no evidence of its functional relevance. Since endocannabinoids and other cannabinomimetics are known to decrease neurogenic contractions in the gut by inhibiting ACh release from enteric neurons[17, 18], we determined whether the atypical cannabinoid, O-1602, considered to be a GPR55 agonist[7–9], can also inhibit the neurogenic contractions in mouse colon. As demonstrated in the representative tracing in Fig. 1A, electrical field stimulation –induced depolarization of enteric neurons produced muscle contractions, due to ACh release from these neurons. The atypical cannabinoid O-1602 inhibited these neurogenic contractions concentration – dependently (Fig. 1A&D).The pD₂ and E_max values of O-1602 for its inhibitory effect were 5.3 ± 0.08 and 89 ± 4%, n=4, respectively. The vehicle, ethanol (even at maximal final concentration of 0.1%), did not affect the electrical field –stimulated contractions (Figure 1B). As a positive control, we confirmed neurogenic contractions by the inhibitory effect of the selective CB1 agonist, WIN 55212-2 on the EFS-evoked contractions (Fig. 1C). O-1602 did not relax the colonic tissues precontracted by high K⁺ (80 mM) (Fig. 1E), confirming the lack of effect of O-1602 on Ca²⁺influx –induced contractions. However, O-1602 (10 μM) but not ethanol has a significant but small (21 ± 3%) inhibitory effect on the exogenous acetylcholine – induced contractions (Figure 1F). Therefore, the site of action of the atypical cannabinoid O-1602 appears to be predominantly presynaptic and partially postjuctional (only at high concentrations).

Figure 1

Effect of the atypical cannabinoid O-1602 on the longitudinal tissue preparations of mouse colon

Inhibitory effect of the atypical cannabinoid O-1602 is independent of CB1- or CB2– but dependent on GPR55 -receptors

We next evaluated whether O-1602-induced functional effects in the mouse colon are mediated through GPR55 receptors. Although the so-called abnormal-cannabidiol receptor antagonist O-1918 [19] possesses GPR55 receptor antagonist properties[20], this compound lacks selectivity as it also antagonizes GPR18[21] and BK(Ca) channels[22]. Therefore, we tested O-1602 over electrical field stimulated –neurogenic contractions in colon tissue strips from GPR55^−/− knock-out mice. O-1602-induced inhibition of neurogenic contractions was significantly reduced in the absence of GPR55 receptors (Fig. 2A). The concentration – response curve of O-1602 in the GPR55^−/− tissue showed a significant rightward shift compared with colon from wild type mice. The pD₂ and E_max values of O-1602 for its inhibitory effect were 5.3 ± 0.08 and 89 ± 4% in wild type; 4.9 ± 0.2 and 53 ± 2% in GPR55^−/−, respectively (n=3-4 in each group). Since some O-1602 effects at higher concentrations appeared to be GPR55-independent, we evaluated the involvement of CB1 and CB2 cannabinoid receptors as well. Therefore, we tested the inhibitory effect of O-1602 over electrical field stimulation – induced neurogenic contractions in colon tissue strips from CB1^−/−/CB2^−/− double knock-out mice. As demonstrated in Fig. 2B, O-1602 maintained its inhibitory actions on neurogenic contractions in spite of the simultaneous absence of both CB1 and CB2 receptors. The concentration –response curves of O-1602 were not significantly different between wild type and the CB1^−/−/CB2^−/− double knock-out mice except at the concentration of 5 μmol/l(Fig. 2C). The pD₂ and E_maxvalues of O-1602 for its inhibitory effect were 5.9 ± 0.1 and 73 ± 4% in CB1^−/−/CB2^−/− double knock-out mice, respectively (n=3-4 in each group). Notably, the CB1^−/−/CB2^−/− double knock-out group shows some enhanced response in relation to wild-type mice at least at the concentration of 5 μmol/l, which could be due to some compensatory effects (enhanced responsiveness of GPR55 receptors) to the lack of both cannabinoid receptors.

Figure 2

Effect of O-1602 on neurogenic contractions in colon from knock-out mice

Inhibitory effect of JWH 015, a CB2/GPR55 agonist on neurogenic contractions in mouse ileum

CB2 receptors have been found predominantly in the peripheral immune system and DRG cells while their role in intestinal contractility is not known in normal mice. Since JWH 015 has inhibitory effects on neurogenic contractions in guinea pig ileum[23], we determined whether GPR55 receptors contribute to the effects of JWH015 in mouse ileum. Compared to the wild type mice, the inhibitory effect of JWH 015 on the neurogenic contractions was not significantly affected by the absence of either CB2 (Fig. 3), GPR55 (Fig. 4A & D), CB1 (Fig. 5B) or CB1/2 (Fig 6A) receptors. Moreover, pharmacological experiments using antagonists of CB1, rimonabant (300 nmol/l) (Fig 5A) or GPR55, cannabidiol (5 μmol/l) (Fig 4C) showed no significant difference compared to the corresponding controls. The respective pD₂ and E_max values of JWH 015 for its inhibitory effect are given in table 1. Since the inhibitory effects of JWH 015 were independent of cannabinoid- and GPR55- receptors, we assessed whether JWH015 directly affects muscarinic –contractions by determining its effects on exogenous acetylcholine-induced concentration–dependent contractions. Preincubation of JWH 015 (3 μmol/l) significantly reduced the E_max of acetyl choline–induced contractions from 107 ± 3% to 67 ± 4%, without affecting pD₂ values (5.8 ±0.1 vs 6.0±0.1; Fig 6B) suggesting an antagonistic effect on muscarinic receptors. However, it is not clear whether the site of action is at receptor level or at some downstream signaling molecule, which needs further investigations.

Figure 3

Effect of JWH015, a CB2/GPR55 agonist, on neurogenic contractions in mouse ileum

Figure 4

Effect of JWH015, a CB2/GPR55 agonist, on neurogenic contractions in ileum from knock-out mice

Figure 5

Concentration –dependent response curves of JWH015, a CB2/GPR55 agonist, on neurogenic contraction inhibition in mouse ileum

Figure 6

A) Concentration –dependent response curves of JWH015, a CB2/GPR55 agonist, on neurogenic contraction inhibition in ileum from wild type (open squares) vs CB1−/−/CB2−/− double knock out (filled squares) mice. B …

Table 1

Potency and efficacy parameters of JWH015 in mouse ileum

Discussion

The salient finding from this study is that we have shown for the first time functional evidence for GPR55- mediated neurogenic effect on intestinal contractility. This conclusion is based on our observation that the atypical cannabinoid, O-1602, a GPR55 agonist, inhibited the neurogenic contractions in the gut and its effects were mediated through GPR55, but neither CB1 nor CB2 receptors were necessary, as evident from corresponding gene knockout mice. The inhibitory effects of O-1602 are predominantly pre-junctional and partially post –junctional (only at high concentrations). Surprisingly, JWH015, considered to be a CB2/GPR55 agonist, inhibits intestinal contractility independently of these receptors.

Two types of cannabinoid receptors: CB1 and CB2[24, 25], which are part of the endocannabinoid system are well studied in several physiological systems. CB1 receptor activation can alleviate pain, reduce GI motility, and affect transient lower esophageal sphincter relaxations and emesis [11, 12]. CB2 receptors are located mainly in immune cells and their activation reduces inflammation. However, there have been several lines of pharmacological evidence supporting the existence of additional putative cannabinoid receptors [4, 26], such as the recently de-orphanized G protein-coupled receptor (GPCR) GPR55 [13, 27, 28]. The functional role of CB1 receptors has been extensively studied in the gastrointestinal tract, however, much less is known about the effects of either CB2 receptors or the newly identified GPR55. Previous reports showed expression of GPR55 receptor in the gut at gene level [13, 16], although contradictory sequences have been reported for hGPR55[5, 29, 30], which could be due to errors in sequencing as alternative splicing is not possible in the intronless GPR55 gene. Therefore, expression of GPR55 at protein level in the system under study becomes critical for further characterization of these receptors. However, the gene expression of GPR55 in the gut suggests a potential role for these orphan GPCRs in gut physiology. Nevertheless, the exact location of GPR55 receptor expression with respect to enteric nervous system, smooth muscle cells, mucosa or other gut cell type remains to be characterized.

The objective of the present study was to elucidate the function of GPR55 receptors in mouse colon using the atypical cannabinoid O-1602, generally considered and used as a pharmacological GPR55 agonist [7–9]. As endocannabinoids, phytocannabinoids and their analogs are known to reduce neurogenic contractions in ileum by suppression of ACh release from enteric neurons [17, 18], we determined whether activation of the GPR55 receptor by O-1602 would also inhibit neurogenic contractions in mouse colon. GPR55 appears to mediate the concentration–dependent inhibitory effect of O-1602 over the electrical field-stimulated contractions because the E_max value of this compound was significantly reduced in colon strips from GPR55^−/− compared to tissue from GPR55^+/+ mice. We also evaluated the involvement of the two accepted cannabinoid receptor subtypes: CB1 and CB2, by testing the O-1602-induced inhibition of neurogenic contractions in colon tissue strips from CB1^−/−/CB2^−/− mice. The finding that O-1602 inhibited the contractions with similar pD₂ and E_max values between double knock-out and wild type mice rules out the involvement of these traditional cannabinoid receptors. The affinity of O-1602 for GPR55 receptors is not clear because EC₅₀ values range from nanomolar to micromolar concentrations and depend on the functional endpoint and cellular systems [6–9]. The data in the present study are consistent with the affinity of O-1602 being in the micromolar range. Although the relatively high concentrations of O-1602 to inhibit contractions raise the likelihood of activating multiple targets, the finding that deletion of GPR55 reduced these effects supports the involvement of these receptors. The use of other GPR55 agonists that possess greater potency and selectivity than O-1602 may help to discern further the physiological function and mechanisms of this receptor. In any event, the results of the present study clearly indicate the actions of O-1602 on the colon are predominantly mediated through GPR55. Although other compounds elicit agonistic effects on GPR55 in vitro such as rimonabant (SR141716A) or AM251 [31], these compounds are not selective. Nonetheless, rimonabant and the reported endogenous ligand for GPR55 lysophosphatidylinositol[32] did not have any effect on the neurogenic contractions in the mouse gut (Fig.5C &D). As of now, further functional characterization of GPR55 receptor is limited by the lack of specific and potent pharmacological modulators. GPR55 is also reported [33] to be activated by the acylethanolamides: palmitoylethanolamide, anandamide and oleoylethanolamide which could also be used for further studies of GPR55 –mediated effects on intestinal motility.

We further examined the mechanisms of the inhibitory action of O-1602 in mouse colon. Even though O-1602 inhibited the neurogenic contractions, the site of action seems to be predominantly prejuctional and partially post-junctional because it also inhibited significantly (at high concentration) the contractions induced by exogenous ACh, suggesting antimuscarinic effects or effects on downstream signaling molecules involved in smooth muscle contractility as well. Depolarization induced by high extracellular K⁺ opens the plasma membrane voltage-gated L-type Ca²⁺ channels, thereby leading to Ca²⁺influx. O-1602 did not affect the high K⁺-induced contraction, ruling out an inhibitory effect on Ca²⁺ influx.

The role of CB2 receptors in the GI tract is uncertain. In an earlier study, the increased GI transit induced by the Gram-negative bacterial endotoxin, lipopolysaccharide, was reduced to normal transit by CB2 agonist. Hence, activation of CB2 receptor in response to lipopolysaccharide is suggested as a mechanism for the re-establishment of normal GI transit after an inflammatory stimulus [34]. Also, JWH015 reduces motility in the croton oil model of intestinal inflammation [35]. Interestingly, hitherto, there is no evidence for any functional role of CB2 receptors in the neurogenic contractions of the normal gut. However, in inflamed gut (ie. LPS –treated), another CB2 agonist, JWH133 reduced the enhanced contractile response in a concentration-dependent manner [36]). Unlike the intestinal preparations, in mouse gastric preparations, JWH015 is demonstrated to inhibit the EFS-evoked cholinergic contractions, which was reduced by AM630, a CB2 antagonist, suggesting a CB2-mediated inhibitory effect [37]. Moreover, CB2 receptors have been found predominantly in the peripheral immune system and DRG cells suggesting no role for CB2 receptors in the regulation of gut motility by enteric neurons. We used JWH 015, considered to be a dual CB2/GPR55 agonist with the twin idea of elucidating the role of each of these receptors in the contractility of intestine. Surprisingly, the inhibitory effects of JWH015 were not at all affected by genetic deletion of either CB2 or GPR55 receptors. Of note, JWH015 has also been shown to mediate its inhibitory effects in guinea pig ileum via CB1 receptors that are sensitive to SR141716A. But, in the present study with the mouse ileum, either pharmacological inhibition of CB1 receptors by SR141716A (rimonabant) or genetic deletion of CB1 receptors, did not affect the JWH015 effects. It has to be noted that the EC₅₀ value of JWH015 is about one order in magnitude higher in mouse ileum (i.e., micromolar range in the current study) than in the guinea pig ileum (i.e., sub-micromolar range)[23]. Interestingly, the same group also found that JWH015 was highly potent in the mouse vas deferentia with an EC₅₀ value in the subnanomolar range; however, these effects were non-CB1 receptor mediated [23]. Thus, the effects of JWH015 appear to be dependent on the system and expression/distribution profile of different cannabinoid receptor subtypes. Since the pD₂ values are much lower and JWH015 effects in the mouse ileum are still present in the various knockout mice used in the present study, its underlying mechanism of action is most likely independent of CB1, CB2 and GPR55 receptors in the mouse gut.

In conclusion, the atypical cannabinoid O-1602, a GPR55 agonist produces its inhibitory effect in the colon via a GPR55 dependent mechanism of action and independent of CB1- or CB2- receptors. Similar to the site of action by established cannabinoids, which mostly occur presynaptically in the gut, the atypical cannabinoid O-1602 also acts predominantly prejunctional and partially postjuctional (at high concentration). The effects of JWH015 in the gut are independent of GPR55, CB1, and CB2 receptors. This study reveals novel functional effects and mechanisms of the atypical cannabinoid O-1602 and GPR55 receptors in the gut, which could have therapeutic benefit in management of intestinal dysmotility in various pathological conditions.

Acknowledgement