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Keywords:

  • cannabinoids;
  • CB receptors;
  • colorectal distension;
  • inflammatory hyperalgesia

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Abstract  Activation of cannabinoid CB1 and CB2 receptors is known to attenuate nociception and hyperalgesia in somatic inflammatory conditions. The aim of this study was to determine whether cannabinoids modulate colonic sensitivity in basal and inflammatory conditions. The effects of CB1 and CB2 receptor agonists and antagonists on the abdominal contractile response to colorectal distension (CRD) in basal conditions and after 2,4,6-trinitrobenzenesulphonic acid-induced colitis were investigated. As previously described, colitis triggered a hypersensitivity to CRD. In basal conditions, both CB1 (WIN 55212-2) and CB2 (JWH 015) agonists reduced the abdominal response to CRD at a dose of 1 mg kg−1, i.p. Both compounds were active at a lower dose (0.1 mg kg−1) abolishing the hypersensitivity induced by colitis. Administered alone, CB1 (Rimonabant) and CB2 (SR 144528) receptor antagonists (10 mg kg−1) had no effect on basal sensitivity. In contrast, the CB1, but not the CB2, receptor antagonist enhanced colitis-induced hyperalgesia. It is concluded that colonic inflammation enhances the antinociceptive action of CB1 and CB2 receptor agonists, and activates an endogenous, CB1 receptor mediated, antinociceptive pathway.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Two cannabinoid receptor subtypes, both G protein-linked, have been isolated and cloned to date. CB1 receptors are mostly expressed by central and peripheral neurones,1 but also by epithelial cells,2 while CB2 receptors are expressed mostly by immune cells.3 Endogenous lipid ligands for these receptors have been isolated and identified. The most known are anandamide4 and 2-arachidonoyl glycerol (2-AG)5,6 that can bind both receptors.

Activation of CB receptors, mainly CB1 receptors, is known to induce a large range of effects on the gastrointestinal tract. For example, CB1 receptor agonists delay gastric emptying in rats,7 inhibit the occurrence of transient lower oesophageal sphincter relaxations (TLOSR) induced by gastric air insufflation in dogs,8 attenuate pentagastrin-induced gastric acid secretion in rats,9 inhibit the electrically evoked acetylcholine and the subsequent contraction in intestinal myenteric plexus-longitudinal muscle preparation in guinea-pig,10 and reduce the ileal secretion induced by electrical stimulation in rats.11 The anandamide congener palmitoylethanolamide inhibits small intestine motility12 and its levels are enhanced in colon biopsies from patients with ulcerative colitis.13

Among the roles of the endocannabonoid system, its analgesic action at spinal and peripheral levels is largely documented.14 However, these data are mainly related to somatic pain and the role of cannabinoids in the control of visceral nociception has been poorly investigated. It has been shown that anandamide, via CB1 receptors, attenuates hyperalgesia-induced urinary bladder inflammation in the rat.15 Concerning the gastrointestinal tract, putative actions of cannabinoids on visceral perception have been proposed,16 but no experimental data are available. Several observations support the hypothesis of a control of gastrointestinal perception by the endocannabinoid system. Anandamide has been found to act at the receptor for capsaicin, the transient receptor potential vanilloid receptor 1 (TRPV1),17 which plays a key role in visceral hyperalgesia. CB1 receptors have been found localized on extrinsic primary afferent nerves in guinea-pig submucosal plexus18 and in the rat dorsal root ganglia.19

Moreover, the role of cannabinoids seems to be enhanced in inflammatory conditions. Besides the anti-inflammatory effects of cannabinoid ligands,20 activation of either CB1 or CB2 receptors is able to attenuate the induction and maintenance of inflammatory pain in rats.21 At the gastrointestinal level, expression of CB1 receptors has been found to be upregulated in rats during an experimental inflammation,22,23 and anandamide levels have been found to be increased in 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis and in ulcerative colitis.24 Consequently, the aim of this study was to determine whether activation of CB1 and CB2 receptors can reduce the sensitivity to colorectal distension (CRD) in the rat and the hyperalgesia induced by TNBS colitis. A role of the endogenous cannabinoid system, in basal and inflammatory conditions, was also investigated by using selective antagonists for CB1 and CB2 receptors.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Animals and surgical procedure

Male Wistar rats (Janvier SA, Le Genest St Isle, France) were surgically prepared for electromyography, according to a previously described technique.25 They were premedicated with acepromazine (0.6 mg kg−1, Calmivet; Vetoquinol, Lure, France) intraperitoneally (i.p.) and anaesthetized by i.p. administration of ketamine (120 mg kg−1, Imalgene 1000; Rhône-Merieux, Lyon, France). Three pairs of nichrome wire electrodes (60 cm in length and 80 μm in diameter) were implanted bilaterally into the abdominal external oblique muscle, 2 cm laterally from the midline, just superior to the inguinal ligament. The free ends of the electrodes were exteriorized on the back of the neck and protected by a glass tube attached to the skin. Rats were individually housed in polypropylene cages and kept in a temperature-controlled room (21 °C). They were allowed free access to water and food (pellets; UAR Epinay, France). All protocols were approved by the Local Animal Care and Use Committee of Institut National de la Recherche Agronomique.

Distension procedure and electromyographic recordings

Rats were placed in polypropylene tunnels (diameter 7 cm, length 20 cm), where they could not move, escape or turn around. They were accustomed to this procedure for 3 days before any CRD, in order to achieve familiarization with that environment.26 The balloon used for CRD was 4 cm long and made from a latex condom. It was fixed on a semi-rigid catheter (2 mm in diameter). CRDs were performed by insertion of the tip of the balloon 4 cm from the anus. The catheter was fixed at the base of the tail and connected to a computerized barostat.27 The balloon was inflated progressively in steps of 15 mmHg, each step of inflation lasting 5 min. Colorectal pressure and balloon volume were continuously monitored on a potentiometric recorder (L6517; Linseis, Selb, Germany) with a paper speed of 1 cm min−1. The electrical activity of the abdominal striated muscle was recorded with an electroencephalograph machine (Mini VIII; Alvar, Paris, France) using a paper speed of 4 cm min−1, and a short time constant (0.03 s) to remove low-frequency signals (<3 Hz).

Experimental protocols

Experiments were performed in groups of eight rats. Colitis was induced in seven groups. After an overnight fast rats were treated with TNBS at a dose of 80 mg kg−1 in 0.3 mL 50% ethanol. TNBS was infused for 30 min through a silicone rubber catheter introduced 3 cm into the anus under acepromazine–ketamine anaesthesia, as described previously.28

Each rat was submitted to CRD 3 days before and 3 days after TNBS administration. Vehicle (10% DMSO/water solution, 0.2 mL) or CB1 and CB2 receptor agonists and antagonists were administered i.p. 20 min before CRD. Group 1 received vehicle, groups 2 and 3 WIN 55212-2 (CB1 receptor agonist) at 0.1 and 1 mg kg−1, respectively, groups 4 and 5 JWH 015 (CB2 receptor agonist) at 0.1 and 1 mg kg−1, respectively, and groups 6 and 7 received at a dose of 10 mg kg−1, Rimonabant (CB1 receptor antagonist) and SR 144528 (CB2 receptor antagonist) respectively. Group 8 was a control group receiving saline instead of TNBS-ethanol, and vehicle of agonists and antagonists. According to the relative low selectivity of the CB1 receptor agonist WIN 55212-2 for CB1 vs CB2 receptors,29 we attempted to block the effects of WIN 55212-2 by the highly selective CB1 and CB2 receptor antagonists, Rimonabant and SR 144528 respectively. Similarly, attempts were made to block the effects of the CB2 agonist JWH 015 with the CB1 and CB2 antagonists. To assess the selectivity of the effects of the CB1 and CB2 receptor agonists these assays were performed in supplementary groups of rats in the absence and presence of colitis, the antagonist being administered i.p. 20 min before the agonist.

Chemicals

WIN 55212-2 [R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]pyrrolo [1,2,3-de]-1,4-benzoazinyl]-(1-naphtalenyl)methanone mesylate] and JWH 015 [(2-methyl-1-propyl1H-indol-3-yl)-1-cyclohexanol] were purchased from Tocris Cookson Inc. (Bristol, UK). Rimonabant [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3- pyrazole-carboxamide] and SR 144528 [N-(1S)-endo-1,3,3,-trimethyl bicyclo [2.2.1]heptan-2yl-5-(4-chloro-3-methyl-phenyl)-1(4-methylbenzyl)- pyrazole-3-carboxamide] were gifts from SANOFI Research Centre (Montpellier, France). All compounds were dissolved in a 10% DMSO/water solution. 2,4,6-trinitrobenzenesulfonic acid was purchased from Sigma (St Louis, MO, USA).

Data analysis

The number of spike bursts (corresponding to abdominal contractions) was determined per 5-min period, from the onset of balloon inflation to the beginning of deflation. Colorectal volumes were determined on a potentiometric recorder as the maximal volume obtained for each stage of distension. Statistical analysis of the number of abdominal contractions and colorectal volumes was performed using a one-way analysis of variance (anova) followed by post hoc test. Data are expressed as mean ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Basal conditions

Under basal conditions (in the absence of inflammation), increasing CRD progressively increased the number of abdominal contractions, starting from 15 mmHg, and these correlated with increases in colonic pressure. Compared with vehicle, both the CB1 receptor agonist, WIN 55212-2, and the CB2 receptor agonist, JWH 015, each at 1 mg kg−1 i.p., significantly reduced (P < 0.05) the number of abdominal contractions evoked during CRD at pressures of 30 and 45 mmHg (Fig. 1). No significant effect (P < 0.05) was observed at the dose of 0.1 mg kg−1 for both compounds (data not shown). Both the CB1 receptor antagonist, Rimonabant, and the CB2 receptor antagonist, SR 144528, at the dose of 10 mg kg−1 i.p., did not modify significantly (P < 0.05) the number of abdominal contractions, whatever the CRD pressure, in comparison with vehicle treatment (Fig. 1). None of the compounds tested modified the balloon volume, irrespective of the distending pressure.

image

Figure 1.  Effect of CB1 (WIN 55212) and CB2 (JWH 015) receptor agonists (1 mg kg−1, i.p.), and CB1 (Rimonabant) and CB2 (SR 144528) receptor antagonists (10 mg kg−1, i.p.) on the number of abdominal contractions induced by increasing pressures of colorectal distensions. *P <  0.05 vs vehicle treatment.

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Inflammatory conditions

Three days after TNBS, the number of abdominal contractions evoked by CRD at the pressure of 15 mmHg was significantly increased (P < 0.05) in comparison with the first CRD session performed 3 days after intracolonic administration of saline (Fig. 2). This increase was abolished by either the CB1 receptor agonist, WIN 55212-2, or the CB2 receptor agonist, JWH 015, at a dose of 0.1 mg kg−1 i.p., which was ineffective in basal conditions. Moreover, the number of abdominal contractions after WIN 55212-2 or JWH 015 was significantly lower (P < 0.05) than in the absence of inflammation for the distending pressure of 30 mmHg (Fig. 2). The CB1 receptor antagonist Rimonabant, at the dose of 10 mg kg−1 i.p., did not affect per se the basal sensitivity, but significantly enhanced (P < 0.05) the increase in the number of abdominal contractions observed at the pressure of 15 mmHg after TNBS. On the contrary, the CB2 receptor antagonist SR 144528, at a dose of 10 mg kg−1 i.p., did not induce any significant change in the number of abdominal contractions after TNBS (P > 0.05), irrespective of the distending pressure (Fig. 2). The balloon volume was significantly reduced (P < 0.05) after TNBS, in comparison with saline, for distending pressures of 45 and 60 mmHg. The compounds tested did not modify these reduced volumes.

image

Figure 2.  Reversal of 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced allodynia by the CB1 receptor agonist (WIN 55212, 0.1 mg kg−1, i.p.) and enhancement by the CB1 receptor antagonist (Rimonabant, 10 mg kg−1, i.p.). The CB2 receptor agonist (JWH 015, 0.1 mg kg−1, i.p.) also reversed TNBS-induced allodynia, but the CB2 receptor antagonist (SR 144528, 10 mg kg−1, i.p.) had no effect. *P <  0.05 vs vehicle, vehicle; †P < 0.05 vs TNBS, vehicle.

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Selectivity of CB1 and CB2 receptor agonists

In the absence of inflammation, the decrease in the number of abdominal contractions induced by the CB1 receptor agonist, WIN 55212-2, at the dose of 1 mg kg−1, was blocked by the CB1 receptor antagonist SR 141718A (10 mg kg−1, i.p.), but not by the CB2 receptor antagonist SR 144528 (10 mg kg−1, i.p.). Conversely, the effect of the CB2 receptor agonist, JWH 015 (1 mg kg−1), was blocked by the CB2 receptor antagonist, but not by the CB1 receptor antagonist (Fig. 3). Identical antagonisms were observed in the presence of colitis, where agonists were tested at the dose of 0.1 mg kg−1 and antagonists at 1 mg kg−1 (Table 1).

image

Figure 3.  Reversal of the effect of the CB1 receptor agonist (WIN 55212) by the CB1 (Rimonabant), but not the CB2 (SR 144528) receptor antagonist. Similarly, the effect of the CB2 receptor agonist (JWH 015) was reversed by the CB2, but not the CB1, receptor antagonist. Agonists and antagonists were administered i.p. at the doses of 1 and 10 mg kg−1 respectively. *P <  0.05 vs vehicle treatment.

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Table 1.   Selective reversal of the effect of WIN 55212-2 by Rimonabant, and JWH 015 by SR 144528, under inflammatory conditions
 VehicleRimonabantSR 144528
  1. Data are reported as number of abdominal contractions during a 5-min period of colorectal distension at 60 mmHg (mean ± SEM, n = 8). Agonists were administered i.p. at the dose of 0.1 mg kg−1, and antagonists at 1 mg kg−1. *P < 0.05 in comparison with agonist/vehicle.

WIN 55212-222.9 ± 3.347.4 ± 4.1*28.2 ± 4.4
JWH 01526.0 ± 3.622.3 ± 3.743.2 ± 4.1*

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Our results indicate that activation of either CB1 or CB2 receptors reduces the basal sensitivity and the colitis-induced hypersensitivity to CRD in rats, both agonists being more active in the presence of colitis. However, the endogenous cannabinoid system is only involved in the inflammatory hyperalgesia and only through CB1 receptors, as the CB1 receptor antagonist, but not the CB2, receptor antagonist, was able to enhance the abdominal response to CRD during colitis.

The decrease in sensitivity to CRD induced by the CB1 receptor agonist is supported by several anatomical data. CB1 receptors are expressed in the gastrointestinal tract, with the highest level in the stomach and in the colon, at least in mice.30 In rats and guinea-pigs, besides an expression in cholinergic myenteric neurones, a close association between CB1 receptor immunoreactivity and fibres labelled for a synaptic protein has been described, suggesting a role in the modulation of transmitter release. Interestingly, very few calcitonin gene-related peptide (CGRP)- and substance P-positive neurones are co-localized with CB1 receptor immunoreactivity, suggesting independent pathways for cannabinoid and CGRP and/or substance P transmission at the digestive level.31 CB1 receptors are expressed in primary sensory neurones of dorsal root ganglia, with some degrees of co-localization with CGRP and TRPV1 ion channel.19,31,32 Finally, CB1 receptors are expressed in the dorsal horn of the spinal cord,33 at both pre- and postsynaptic levels.34

CB2 receptors are known to be mostly located on immune cells, and there is no clear anatomical evidence to support the decrease in sensitivity to CRD by the CB2 receptor agonist in basal conditions. In humans, CB2 receptors have been identified in normal colonic epithelial cells,2 as well as in colonic tissues of inflammatory bowel diseases35 and colorectal carcinomas.2 CB2 receptors have been also found on skin afferent nerve fibres,36 but there is no evidence for CB2 expression in gastrointestinal afferents. No CB2 receptor has been identified in the mouse dorsal root ganglia or spinal cord, in basal conditions.37 However, there are some data available indicating an antinociceptive action of CB2 receptor activation. An analgesic action of CB2 receptor agonists has been shown in inflammatory or neuropathic pain, but only few data provide evidence for these compounds in basal conditions. A CB2 receptor-selective agonist, AM 1241, produces antinociception to skin thermal stimuli.38 The same compound was also able to suppress C-fibre activity in the absence of inflammation.39

We found an enhanced activity of both CB1 and CB2 receptor agonists after induction of colitis. A colonic anti-inflammatory action of CB1 receptor has been suggested. Expression of CB1 receptors has been found to be upregulated in rats during an experimental intestinal inflammation.22,23 CB1-deficient mice or wild-type mice treated with the CB1 receptor antagonist Rimonabant display a stronger experimental colitis than controls.23 At the somatic level, the endocannabinoid palmitoylethanolamide and the synthetic cannabinoid nabilone are able to reduce a carrageenan-induced acute hindpaw inflammation.40 However, the anti-inflammatory action of cannabinoid is not well established, and a pro-inflammatory role has been suggested. The CB1 receptor antagonist Rimonabant has been found to reduce an intestinal inflammation induced by indomethacin or lipopolysaccharide (LPS).41 Similarly, intraluminal administration of anandamide or 2-AG causes an iletis in the rat.42 Nevertheless, the anti-hyperalgesic action of CB1 or CB2 receptor agonists that we observed after TNBS treatment cannot be attributed to a possible anti-inflammatory action, according to the short delay (20 min) between administration of the compounds and the measurement of colonic sensitivity to distension.

An action of both CB1 and CB2 receptor activation in inflammatory conditions, at least at the somatic level, has been largely documented.14 The excitatory potency of anandamide on TRPV1 receptor is increased by inflammatory mediators such as bradykinin or prostaglandin E2, but the consequence in terms of hyperalgesia or hypoalgesia has not been established.43 This is in agreement with other data indicating a pronociceptive or an antinociceptive effect of cannabinoids in inflammatory conditions. Local administration of anandamide increases c-fos expression in the rat inflamed urinary bladder and contributes to the development of hyperalgesia during cystitis.44 On the contrary, peripheral activation of either CB1 or CB2 receptors has been found to suppress spinal c-fos expression induced by intraplantar carrageenan.45 Other studies clearly indicate an antinociceptive effect of CB1 or CB2 activation in inflammatory hypersensitivity.39,46 We have shown that the efficacy of CB1 and CB2 agonists was greater in reducing the abdominal response to CRD during colitis than in the basal state. Interestingly, a similar enhanced activity has been described for the CB2 receptor agonist AM 1241 in reducing C-fibre activity induced by stimulation of carrageenan-inflamed paw, in comparison with the non-inflamed paw.39 At the level of the gastrointestinal tract, the effects of CB receptor agonists have been found enhanced in inflammatory conditions. For example, the CB1 receptor agonist CP 55940 is more active in delaying intestinal transit in croton oil-treated mice, where CB1 receptors have been found to be upregulated, than in controls.22 Similarly, the CB2 receptor agonist JWH 133 increases gastrointestinal transit in LPS-treated rats, but does not modify basal transit.47

Our results indicate that endogenous cannabinoids are involved through CB1 receptors as a feedback mechanism limiting colitis-induced hypersensitivity, as in these conditions the CB1 receptor antagonist Rimonabant administered alone enhances the abdominal response to CRD. This is in agreement with a recent finding showing that anandamide, but not 2-AG, levels are strongly increased in the colon submucosa of TNBS-treated rats and in biopsies of patients with ulcerative colitis.24 This increase in anandamide level, which displays a very low efficacy at CB2 receptors,29 and the upregulation of CB1 receptors during intestinal inflammation,22,23 may explain the effect of the CB1 receptor antagonist in inflammatory conditions. On the other hand, it has been recently shown in mice that activation of TRPV1 receptors reduces the inflammation-induced irritation of colonic smooth muscle activities.48 It can be speculated that the abdominal response to CRD after TNBS may be modulated through TRPV1 receptors in two ways. If the elevated anandamide levels associated with TNBS-induced colitis still activate TRPV1 receptors when CB1 receptors are blocked, the enhancement of the abdominal response to CRD induced by Rimonabant may be underestimated. On the contrary, high doses of Rimonabant have been found to inhibit some TRPV1-mediated effects of anandamide.49 A blockade of TRPV1 receptors by Rimonabant may explain the enhanced abdominal response.

Using the antagonist Rimonabant, an involvement of CB1 receptors has also been shown in the regulation of gastrointestinal functions, in the absence of inflammation. For example, Rimonabant enhances the cholinergic and NANC contractile response of the guinea-pig ileum elicited by electrical stimulation,50 but does not affect the response of the human ileum.51 It increases defecation and accelerates upper gastrointestinal transit in basal conditions in rats,52 and increases the occurrence of TLOSR induced by gastric air insufflation in dogs.8 On the contrary, it does not modify gastric emptying in rats.7 The endogenous involvement of CB1 receptors in the attenuation of hyperalgesia to CRD during TNBS-induced colitis is in agreement with their overexpression observed in the same inflammatory model, at least on myenteric neurones,23 and in croton oil-induced inflammation.22 Interestingly, CB1 receptors have been found to be expressed and functional in mast cells, which are involved in several models of visceral hyperalgesia.53 Moreover, nerve growth factor (NGF) is known to play a key role in the colonic inflammatory hyperalgesia,54 and to maintain visceral hyperalgesia over long periods.55 CB1 receptors are expressed in NGF-responsive neurones, but their expression is not regulated by NGF.56 On the contrary, visceral hyperalgesia induced by NGF (local application of NGF in the urinary bladder) is attenuated by activation of CB1 receptors.57 Another possible link between CB1 receptors and inflammatory nociception can be found at the level of afferent colonic neurones expressing TRPV1 receptors, which are sensitized by inflammatory mediators.58 CB1 and TRPV1 receptors have been found co-localized in rat dorsal root ganglia,19 anandamide is able to act at TRPV1 receptors,17 TRPV1 receptors have been proposed as a molecular target for the antihyperalgesic action of CB1 agonists in a rat model of acute somatic inflammation,59 and CB1 receptor antagonists have been found to inhibit some TRPV1 receptor-mediated effects of anandamide.49

Finally, our results have shown an enhanced activity of CB1 and CB2 receptors, and an endogenous involvement of CB1 receptors in the attenuation of inflammatory colonic hyperalgesia, but do not permit to identify a site of action. A body of evidence suggests an action at the level of afferent neurones, dorsal root ganglia or dorsal horn of the spinal cord, but an action at the level of the central nervous system cannot be excluded.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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