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Keywords:

  • cannabinoid receptors;
  • gastrointestinal motility;
  • radiology;
  • rat

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing Interests
  8. References

Background  In the absence of pathology, cannabinoid-induced depression of gastrointestinal (GI) motility is thought to be mediated primarily by CB1 receptors, whereas the role of CB2 receptors is still unclear. The aim of this work was to radiographically analyze the acute effect of the mixed cannabinoid agonist WIN 55,212-2 (WIN) on GI motor function in the rat, focusing on the involvement of CB1 and CB2 receptors.

Methods  Male Wistar rats received different doses of WIN and both psychoactivity (cannabinoid tetrad) and GI motility (radiographic analysis) were tested. The duration of WIN effect on GI motility was also radiographically analyzed. Finally, the involvement of the different cannabinoid receptors on WIN-induced alterations of GI motility was analyzed by the previous administration of selective CB1 (AM251) and CB2 (SR144528 or AM630) antagonists. After administration of contrast medium, alterations in GI motility were quantitatively evaluated in serial radiographs by assigning a compounded value to each region of the GI tract.

Key Results  Low, analgesic doses of WIN delayed intestinal transit, but high, psychoactive doses were required to delay gastric emptying. Acute WIN effects on GI motility were confined to the first few hours after administration. AM251 partially counteracted the effect of WIN on GI motility. Surprisingly, SR144528 (but not AM630) enhanced WIN-induced delayed gastric emptying.

Conclusions & Inferences  X-ray analyses confirm that cannabinoids inhibit GI motility via CB1 receptors; in addition, cannabinoids could influence motility through interaction with a SR144528-sensitive site. Further studies are needed to verify if such site of action is the CB2 receptor.

Marijuana (Cannabis sativa) has been used for recreational and medicinal purposes for centuries. Natural derivatives and synthetic analogues, known as cannabinoid drugs, result in a number of central and peripheral effects. In the gastrointestinal (GI) tract, cannabinoids exert mainly an inhibitory role, reducing motility and secretion in both physiological and pathophysiological conditions.1–5

After the discovery and cloning of the cannabinoid receptors,6 the CB1 receptor emerged as the primary cannabinoid receptor expressed in both the central and peripheral nervous systems, including the enteric nervous system (ENS) of the gut. In contrast, CB2 receptor expression was thought to be restricted to cells and organs of the immune system.6,7 However, recent data have shown that CB1 receptors are also present on non-neural tissues, such as liver and adipose tissue, and CB2 receptors are expressed in the brain, and in the periphery.2,8–10

The in vivo and in vitro depressive effects of cannabinoids on GI motor function have been mostly attributed to CB1-mediated actions of these drugs on presynaptic nerve terminals of the myenteric motor neurones innervating the muscle and leading to reduced neurotransmitter release, namely acetylcholine.1,3,5 In contrast, a functional role for CB2 receptors in normal animals has been, thus far, difficult to prove.11 CB2 receptor expression has been observed in the ENS in rodents,12 and it is also present on enteric neurons of the human ileum.11 In the rat, mRNA for these cannabinoid receptors has been isolated from both full-wall thickness preparations of the stomach and dissected preparations of the ileum containing only longitudinal muscle with the adherent myenteric plexus (LMMP).13 In spite of this evidence, most studies report no effect of CB2 receptor activation on GI motility under physiological conditions.14,15 It is now generally thought that a role for CB2 receptors in the control of GI motility could indeed exist, but only in some regions of the gut, namely the stomach, or under some specific pathophysiological states (e.g., inflammatory, visceral pain conditions).11,12

Non-invasive radiographic techniques allow for the analysis of the effect of a drug on different GI regions at different time points.16,17 Alterations on gastric motor function (i.e., gastric emptying delay, gastric distension) are particularly well detected by these means. Using these techniques, we have recently confirmed that the mixed cannabinoid agonist WIN 55,212–2 (WIN), acutely or chronically administered, depresses GI motility. Whereas both psychoactive and non-psychoactive doses depressed intestinal transit, gastric emptying was delayed only after administration of a high, psychoactive dose. After daily WIN administration, gastric emptying was still significantly delayed, but this effect was sensitive to CB1 receptor blockage.17 However, the role of CB2 receptors was not tested. Therefore, the aim of the present study is to extend these observations by further analyzing dose-dependency, duration and involvement of CB1 and CB2 receptors in the acute effect of WIN on GI motor function in the rat.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing Interests
  8. References

The experiments were designed and performed in strict accordance with the EC regulations for care and use of experimental animals (EEC Nº 86/609), and were approved by the Ethical Committee at the Universidad Rey Juan Carlos.

Animals

Male Wistar rats (240–300 g) were obtained from Harlan-Iberica (Barcelona, Spain), and were housed (4–6 per cage) in standard transparent cages (60 cm × 40 cm × 20 cm) that were furnished with wood shaving bedding, which was changed every 1–2 days. Cages were placed adjacent to each other under environmentally controlled conditions (temperature = 20 °C; humidity = 60%) with a 12 h light/12 h dark cycle (lights on between 08:00 and 20:00 h). Animals had free access to standard laboratory rat chow (Harlan-Iberica) and tap water.

Drug treatments

One week after the animals arrived at the laboratory, three different sets of experiments were carried out. In the first one, rats received an intraperitoneal (i.p.) injection of saline (0.9% NaCl weight/volume), vehicle (see below) or WIN (0.5, 1, 2 and 5 mg kg−1), and either psychoactive effects or alterations in GI motility were measured (see below). In the second set of experiments, rats received vehicle or WIN at 5 mg kg−1 (WIN 5), and GI motility was radiographically analyzed for 24 h, starting immediately after administration (day 1) or the following day (day 2), in order to detect long-lasting effects of WIN. We also tested whether there remained signs of psychoactivity 24 h after administration of WIN at 5 mg kg−1. Finally, in the third set of experiments, the role of CB1 and CB2 receptors on the effect of WIN on GI motility was also radiographically determined by administering either the CB1 antagonist AM251 (1 mg kg−1, i.p.17), the CB2 antagonist SR144528 (1 mg kg−1, i.p.18) or both antagonists 30 min prior to WIN 5. The effect of each antagonist or the combination of both antagonists was also analyzed in vehicle-treated rats. In all experiments, drug volumes were adjusted to a maximum of 4–5 mL kg−1. The effect of a second CB2 antagonist, AM630 (1 mg kg−1, i.p.19), was also tested both alone and in combination with WIN.

Central effects of WIN 55,212-2 (cannabinoid tetrad)

The classical cannabinoid tetrad consists of the combination of four tests that evaluate temperature, antinociception, catalepsy, and motility in the same animal after cannabinoid administration,20 and was used to identify non-psychoactive and psychoactive doses of WIN. The test values were recorded by an observer unaware of the treatments, as previously reported.17

Heat-antinociception was tested 20 min after drug administration using a 37370 plantar test apparatus (Ugo Basile, Comerio VA, Italy). The withdrawal latency from a focused beam of radiant heat applied to the mid plantar surface of the hindpaws was recorded. The intensity of the light was adjusted at the beginning of the experiment so that the control average baseline latencies were about 8 s and a cut-off latency of 25 s was imposed. The withdrawal latency of each paw was measured during three trials separated by 2 min intervals, and the mean of the three readings was used for data analysis.

To measure catalepsy, the rats were hung by their front paws from a rubber-coated metal ring (12 cm diameter) fixed horizontally at a height that allowed their hindpaws to just touch the bench. The time taken for the rat to move off the ring was measured with a cut-off limit of 30 s. Latencies were measured 25 min after drug or vehicle administration.

Rectal temperature was recorded 30 min after drug administration using a P6 thermometer and a lubricated rectal probe (Cibertec S.A., Madrid, Spain) inserted into the rectum to a constant depth of 5 cm.

Spontaneous locomotor activity was evaluated using individual photocell activity chambers (Cibertec S.A.). Rats were placed in the recording chambers (55 × 40 cm, with a 3-cm spacing between beams) 40 min after drug administration; the number of interruptions of photocell beams was recorded over a 10-min period. The mean number of crossings of the photocell beams was used for comparison.

Evaluation of GI motor function

Gastrointestinal motor function was studied by radiographic methods previously described.16,17 Immediately after drug injection (or 24 h after, in some experiments), 2.5 mL of a suspension of barium sulfate (Barigraf®, 2 g mL−1, t° = 22 °C) were administered per os. Plain facial radiographs of the GI tract were performed using a Helident DS X-ray apparatus (Sirona Iberica, Barcelona, Spain; 60 kV, 7 mA) with a focus distance manually fixed to 50 ± 1 cm. Exposure time was adjusted to 0.06 s. Immobilization of the rats in the prone position was achieved by placing them inside hand-made transparent plastic tubes, which were adjusted to the size of the rat so that they could not move, escape or turn around. Habituation to the recording chamber prior to commencement of the study did not significantly alter GI motility.16 In order to further reduce stress, rats were released immediately after each shot (immobilization lasted for 2–3 min). X-rays were recorded on Kodak Ektavision G film (15 × 30 cm) housed in a hand-made cassette provided with regular intensifying screen at different times (immediately and 1, 2, 4, 6, 8 and 24 h: T0–T24) after administration of the contrast medium. The film cassette was located directly beneath the restraining tube. While taking the radiographs, the qualified investigator remained at least 2 m away from the X-ray source, where radioactivity while shooting was not different from environmental readings. Films were developed in a Kodak X-omat 2000 automatic processor. Analysis of the radiographs was performed by a trained investigator blind to the drug administered. Alterations in GI motility were semi-quantitatively determined from the images at each time-point as previously described,16 by assigning a compounded value (from now on referred to as ‘contents’) to each region of the GI tract, from 0 to 12 points, considering the following parameters: percentage of the GI region filled with contrast (0–4); intensity of contrast (0–4); homogeneity of contrast (0–2); and sharpness of the GI region profile (0–2). Qualitative differences in gastric and intestinal shape, size, tone, and peristaltic activity were also recorded.

Compounds and drugs

Barium sulfate (Barigraf® AD; Juste SAQF, Madrid, Spain) was suspended in tap water and continuously hand-stirred until administration. WIN was obtained from Tocris Cookson (Bristol, UK) and dissolved in ethanol 1 mg : 1 mL and, subsequently, in ethanol and Tween 80 (1 : 2), after which, the ethanol was evaporated and saline added to reach the final concentration.21 AM251 was obtained from Ascent Scientific (North Somerset, UK). SR144528 was kindly gifted by Sanofi-Aventis Recherche & Developpement (Montpellier, France). AM630 was purchased from Tocris Cookson (Bristol, UK). The three antagonists were dissolved as described for WIN.

Statistical analysis

Data are presented as the mean ± SE. Differences between groups were analyzed using unpaired Student’s t-test, with Welch’s correction where appropriate or one- or two-way anova followed by post hoc Bonferroni multiple comparison test. Values of P < 0.05 were regarded as being significantly different.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing Interests
  8. References

Dose-dependency of central and gastrointestinal effects induced by WIN 55,212-2

Four different doses of WIN were tested for psychoactivity, using the cannabinoid tetrad (Fig. 1), and for effects on GI motility (Fig. 2). Values obtained for saline- and vehicle-treated rats were not significantly different. The lowest dose of WIN tested (0.5 mg kg−1), induced significant analgesia, but none of the remaining signs of the tetrad (Fig. 1). It did not alter gastric emptying either (Fig. 2A), but slightly delayed intestinal transit, which was measured as arrival and filling of the cecum (Fig. 2C). Arrival and filling of the colorectum was also delayed but probably only as a consequence of delayed transit in the cecum (Fig. 2D).

image

Figure 1.  Dose-dependency of acute central effects of the cannabinoid agonist WIN 55,212-2. Cannabinoid central (psychoactive) effects were evaluated using the cannabinoid tetrad: (A) plantar test (for heat-analgesia); (B) ring test (for catalepsy); (C) rectal temperature (for hypothermia); (D) spontaneous locomotor activity (for hypolocomotion). Rats were injected intraperitoneal (i.p.) with: saline (4–5 mL kg−1, n = 12), vehicle (n = 12) or WIN 55,212-2 (WIN) at 0.5 (WIN 0.5, n = 12), 1 (WIN 1, n = 4), 2 (WIN 2, n = 4), or 5 mg kg−1 (WIN 5, n = 12). Bars show mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle (unpaired Student’s t-test with Welch’s correction).

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image

Figure 2.  Dose-dependency of acute effects of the cannabinoid agonist WIN 55,212-2 on gastrointestinal motor function. Gastrointestinal motor function was evaluated by radiological methods (see text) in: (A) stomach (gastric emptying); (B) small intestine; (C) cecum; (D) colorectum. Rats were injected intraperitoneal (i.p.) with: saline (4–5 mL kg−1, n = 12), vehicle (n = 8) or WIN 55,212-2 (WIN) at 0.5 (WIN 0.5, n = 12), 1 (WIN 1, n = 12), 2 (WIN 2, n = 16), or 5 mg kg−1 (WIN 5, n = 18). Barium sulfate (2.5 mL, 2 g mL−1) was intragastrically administered immediately after drug administration. Data represent mean ± SE. P < 0.05, for WIN 2 vs saline; $$P < 0.01, for WIN 0.5, #P < 0.05, ##P < 0.01, for WIN 1, +++P < 0.001, for WIN 2 and ***P < 0.001, for WIN 5 vs vehicle (two-way anova). For WIN 2, the results of the Bonferroni post hoc test (not shown in the figure, for more clarity) were: for the stomach, T1, P < 0.05; T2, P < 0.001, vs saline-treated animals; for the small intestine, T1: P < 0.01, T4: P < 0.05, T6: P < 0.001 and T8: P < 0.01 vs vehicle; for the cecum, T2: P < 0.001, T4: P < 0.001 vs vehicle; for the colorectum, T4: P < 0.001, T6: P < 0.001 vs vehicle. (E) X-rays from rats treated with vehicle (VEH) or WIN 2; radiographs were taken 0, 1, 2 and 4 h after drug administration. Scale bar: 4 cm.

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The results obtained with the dose of 1 mg kg−1 in both the cannabinoid tetrad (Fig. 1) and the different GI regions (Fig. 2) were practically identical to those obtained with 0.5 mg kg−1.

The dose of 2 mg kg−1 was the first one to induce more than one sign in the tetrad. Thus, analgesia (Fig. 1A) and catalepsy (Fig. 1B) were clearly present. Spontaneous locomotor activity was also reduced, although the difference did not reach statistical significance (Fig. 1D). Hypothermia was absent (Fig. 1C). With regards to GI motility, also both gastric emptying (Fig. 2A) and transit in all intestinal regions (Fig. 2B–D) was significantly delayed. The delaying effect of this dose was higher than that induced by the previous one (1 mg kg−1) in the cecum (Fig. 2C), but similar in the colorectum (Fig. 2D).

Finally, WIN 5 induced the four signs of the tetrad (Fig. 1), and also induced a significant delay in gastric emptying (Fig. 2A) and intestinal transit (Fig. 2B–D).

Duration of WIN 55,212-2 effects on gastrointestinal motor function and in the cannabinoid tetrad

Vehicle-injected rats showed similar motility in all GI regions irrespective of when (day 1 or day 2) barium was administered; therefore, to simplify, in Fig 3 the mean curve of both days for each region is shown. As in the control, in the group treated with WIN 5, at T24 during day 1 contrast medium was practically absent from all GI regions (Fig. 3, day 1). When the contrast medium was given 24 h after administration (day 2), gastric emptying and intestinal transit were practically identical in vehicle- and WIN-treated animals (Fig. 3). Finally, WIN at 5 mg kg−1 did not exert any significant psychoactive effect when tested 24 h after its administration and values were as in control (saline- or vehicle-treated) rats.

image

Figure 3.  Duration of the effect of the cannabinoid agonist WIN 55,212-2 on gastrointestinal motor function in the rat. Gastrointestinal motor function was evaluated by radiological methods (see text) in: (A) stomach (gastric emptying); (B) small intestine; (C) cecum; (D) colorectum. Rats were injected intraperitoneal (i.p.) with: vehicle (n = 12) or WIN 55,212-2 (WIN) at 5 mg kg−1 (WIN 5) on day 1. Barium sulfate (2.5 mL, 2 g mL−1) was intragastrically administered immediately after WIN 5 (WIN 5-day 1; n = 18) or 24 h after (WIN 5-day 2; n = 4). Data represent mean ± SE. *P < 0.05, ***P < 0.001 vs vehicle; ##P < 0.01, ###P < 0.001 vs WIN 5-day 1 (two-way anova followed by Bonferroni post hoc test). (E) X-rays from rats treated with WIN 5; barium was administered immediately (day 1) or 24 h after WIN (day 2), and radiographs were taken 0, 2, 8 and 24 h after barium administration. Scale bar: 4 cm.

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Involvement of CB1 and CB2 receptors in the GI effects induced by WIN 55,212-2

As shown in Fig. 4, the effect of WIN 5 was prevented at least partially, by prior administration of the CB1 antagonist. Thus, after WIN administration, in those animals that had also received AM251, gastric emptying seemed to be delayed for the first 2 h (as in animals treated only with WIN), but thereafter gastric emptying was more similar to vehicle-treated rats (Fig. 4A). In the small intestine, the filling phase was shorter (2 h) than that obtained for WIN-treated rats (4–6 h), whereas the emptying phase overlapped with that from controls (Fig. 4B). The motility curve for the cecum was significantly displaced to the left, but did not overlap with that obtained from control rats (Fig. 4C). Finally, in the colorectum, the effect of WIN was completely prevented by AM251 (Fig. 4D). As compared with vehicle-treated animals, AM251 alone did not modify the different motility curves, except for that of colorectum, in which filling was slightly but significantly accelerated (Fig. 4D).

image

Figure 4.  Effect of the cannabinoid CB1 antagonist AM251 on gastrointestinal motor function in the rat. Gastrointestinal motor function was evaluated by radiological methods (see text) in: (A) stomach (gastric emptying); (B) small intestine; (C) cecum; (D) colorectum. Rats were injected intraperitoneal (i.p.) with: vehicle (n = 12), WIN 55,212-2 (WIN) at 5 mg kg−1 (W5, n = 18), AM251 at 1 mg kg−1 (AM, n = 12) or both WIN and AM (AM + W5, n = 8). Barium sulfate (2.5 mL, 2 g mL−1) was intragastrically administered immediately after drug administration. Data represent mean ± SE. ***P < 0.001 vs vehicle; ###P < 0.001 vs W5 (two-way anova followed by Bonferroni post hoc test). (E) X-rays from rats treated with vehicle (VEH), W5, AM + W5 or AM; radiographs were taken 6 h after barium administration. Scale bar: 4 cm.

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When the CB2 antagonist SR144528 was administered prior to WIN, the motility curve for gastric emptying was unexpectedly delayed for even longer (Fig. 5A). Probably due to these gastric effects, the motility curves for the small intestine (Fig. 5B), cecum (Fig. 5C) and colorectum (Fig. 5D) were also significantly displaced to the right, and filling of these regions started at least 2 h later than in WIN only treated rats. SR144528 did not induce any significant effect on GI motility when given alone (Fig. 5). In view of the effects of SR144528, a second CB2 antagonist, AM630, was tested. However, AM630 neither significantly altered GI motility when given alone nor significantly modified WIN depressive effect.

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Figure 5.  Effect of the cannabinoid CB2 antagonist SR144528 on gastrointestinal motor function in the rat. Gastrointestinal motor function was evaluated by radiological methods (see text) in: (A) stomach (gastric emptying); (B) small intestine; (C) cecum; (D) colorectum. Rats were injected intraperitoneal (i.p.) with: vehicle (n = 12), WIN 55,212-2 (WIN) at 5 mg kg−1 (W5, n = 18), SR144528 at 1 mg kg−1 (SR2, n = 8) or both WIN and SR2 (SR2 + W5, n = 8). Barium sulfate (2.5 mL, 2 g mL−1) was intragastrically administered immediately after drug administration. Data represent mean ± SE. ***P < 0.001 vs vehicle; #P < 0.05, ##P < 0.01, ###P < 0.001 vs W5 (Two-Way anova followed by Bonferroni post hoc test). (E) X-rays from rats treated with vehicle (VEH), W5, SR2 + W5 or SR2; radiographs were taken 8 h after barium administration. Scale bar: 4 cm.

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When both AM251 and SR144528 were administered before WIN, the motility curves for all regions were not significantly different from those obtained in vehicle-treated animals (Fig. 6). When given without WIN, the combination of antagonists slightly, but significantly, accelerated transit in the intestine, particularly in the cecum and the colorectum (Fig. 6C,D).

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Figure 6.  Effect of the combination of both cannabinoid CB1 (AM251) and CB2 (SR144528) antagonists on gastrointestinal motor function in the rat. Gastrointestinal motor function was evaluated by radiological methods (see text) in: (A) stomach (gastric emptying); (B) small intestine; (C) cecum; (D) colorectum. Rats were injected intraperitoneal (i.p.) with: vehicle (n = 8), WIN 55,212-2 (WIN) at 5 mg kg−1 (W5, n = 18), AM251 (AM) and SR144528 (SR2) at 1 mg kg−1 alone (AM + SR2, n = 8) or both antagonists together with WIN (AM + SR2 + W5, n = 8). Barium sulfate (2.5 mL, 2 g mL−1) was intragastrically administered immediately after drug administration. Data represent mean ± SE. **P < 0.01, ***P < 0.001 vs vehicle; ##P < 0.01, ###P < 0.001 vs WIN 5 (two-way anova followed by Bonferroni post hoc test). (E) X-rays from rats treated with vehicle (VEH), W5, AM + SR2 + W5 or AM + SR2; radiographs were taken 4 h after barium administration. Scale bar: 4 cm.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing Interests
  8. References

Non-invasive radiographic techniques were used to analyze the acute effect of the cannabinoid agonist WIN 55,212-2 on GI motility in the rat. It was confirmed that: low (although analgesic) doses are capable of reducing intestinal transit, whereas gastric emptying is delayed by higher (psychoactive) doses; acute WIN administration does not exert long-lasting effects on GI motility or in the cannabinoid tetrad; the CB1 receptor is involved in reducing GI motility. Interestingly, the CB2 receptor antagonist SR144528 did not block but enhanced WIN-induced delayed gastric emptying, suggesting that not only CB1 receptors but also a SR144528-sensitive site (CB2 receptors?) might be involved in this effect, with differential roles. Thus, this SR144528-sensitive site might function as a brake to the delaying effect of CB1 activation.

Dose-dependency and duration of WIN 55,212-2 effects

As previously shown,17 psychoactive doses of WIN induced a delay in gastric emptying. In our hands, the dose of 2 mg kg−1 is the minimum required to induce significant catalepsy,22,23 which is sometimes chosen as an isolated indicator of psychoactivity,24 and is also capable of delaying gastric emptying in the rat. Thus, in the rat, catalepsy and delayed gastric emptying require similar, high, cannabinoid doses to be induced, whereas analgesia and intestinal motility impairment need lower doses. These differences might be related to the receptors involved, since in the intestine, in the absence of pathology, the cannabinoid effect is likely mediated only by CB1 receptors, whereas in the stomach CB2 receptors might also play a functional role (see below). Interestingly, in the central nervous system, CB2 receptors were also recently shown to induce some behavioral effects, including catalepsy at high doses.25,26

Although WIN has a relatively long half-life (24–36 h27,28), and high doses might induce central effects for several hours,29 no residual sign of psychoactivity was found when the cannabinoid tetrad was performed 24 h after WIN administration. Similarly, its acute effects on GI motor function seemed to be restricted to the first 2–6 h after administration, with normal motility being reached immediately afterwards (present results,17). Nevertheless, due to the movement of contrast medium towards the aboral end of the GI tract throughout the experiment, long-lasting effects (i.e., gastric distension16) might have resulted undetected. Therefore, medium contrast was administered 24 h after WIN administration. In this experiment, the motility curves for the different GI regions were comparable to the ones obtained from vehicle-treated rats and no gastric distension was apparent (see Fig. 3E), further confirming the relatively short-lasting effect of WIN on GI motility.

Involvement of CB1 receptors in the effect of WIN 55,212-2 on GI motility

Our results, using radiographic, non-invasive methods for evaluating GI motor function, are in agreement with numerous studies showing that cannabinoids inhibit contractility in vitro and motility in vivo mainly via CB1 receptors.3,5,17,30,31 However, although AM251-induced blockage of WIN effect was complete for the small intestine (emptying phase) and the colorectum, it was only partial in the stomach and the cecum. In these regions, higher doses of AM251 might be necessary to completely counteract WIN effect, but the same dose tested here (1 mg kg−1) prevented the remaining effect of the agonist on GI motility (particularly gastric emptying) after chronic treatment.17 Alternatively, other receptors might be involved in WIN effect (see below). In these regards, when both CB1 and CB2 antagonists were given in combination, the effect of WIN was completely prevented in all GI regions. These results support the involvement of CB2 receptors in WIN-induced GI effects.

CB1 antagonists, including AM251,32 might exert an inverse agonist action or unmask an endogenous cannabinoid inhibitory tone in the GI tract.33–38 Indeed, when given alone, AM251 exerted a slight, but significant accelerating effect in the colorectum (and in the cecum when it was combined with SR144528).

Involvement of CB2 receptors in the effect of WIN 55,212-2 on GI motility

The CB2 antagonists SR144528 and AM630, at the same dose used for the CB1 antagonist AM251, did not block WIN effect in the stomach or the intestine. On the contrary, SR144528 significantly enhanced WIN-induced delayed gastric emptying. In contrast to SR144528, AM630 did not increase WIN effect, but this might be due to the fact that AM630 shows a lower affinity for CB2 receptors than SR144528 (Ki = 31.2 nmol L−1vs, 0.28 to 5.6 nmol L−1, respectively39).

In most in vivo studies, SR144528 did not modify cannabinoid-induced delayed gastric emptying.18,40,41 The discrepancy between these and our results might be due to the different methodologies utilized. In fact, those studies were carried out within the first hour after WIN administration, using invasive techniques, whereas our radiographic methods are non-invasive, and alterations in motility curves of all GI regions are best detected 2–8 h after drug administration. Although gastric emptying was already reduced 1 h after administration of WIN at 5 mg kg−1 (at that time point very little contrast medium was found in the small intestine and the corresponding motility curve was already significantly altered: present results,17), the slight increase in the effect of WIN in the presence of SR144528 was significant only at least 2 h after drug administration.

The effects of SR144528 on gastric emptying could have been mediated through CB2 receptors located in the stomach. Thus, mRNA was isolated from full-wall thickness preparations of rat esophagus and stomach.13 Whereas the effect of cannabinoids on altering nonadrenergic noncholinergic relaxation seems to depend on the species and/or the preparation used,13,42 gastric cholinergic contractions elicited in vitro were depressed by cannabinoids,13,42 and AM630 blocked this effect at least in the isolated murine stomach.42 Despite these in vitro evidences, it has been difficult to demonstrate an in vivo effect of CB2 receptors. Our results suggest that longer contact times or stronger CB2 manipulation (higher doses and/or higher affinity of the antagonist for the CB2 receptors) might be required to unmask in vivo actions of cannabinoids on CB2 receptors. In addition, the actions at the nerve terminals, which are those analyzed in vitro, might be counteracted by actions at other levels (central, vagal, other GI regions such as the pylorus…). In fact, we can not discard the involvement of central pathways mediating WIN effect (with participation of some CB2 receptors?), since only psychoactive doses were capable of effectively reducing gastric emptying (present results,17). Also, CB2-mediated actions might be unmasked under certain (pathological) situations, like in the intestine.11,12 Finally, since AM630, the other CB2 antagonist used here, did not modify the effect of WIN, it can not be discarded that SR144528 might have acted upon a different (not yet identified) site.

It is not clear why SR144528 further delayed gastric emptying. SR144528 could have exerted a direct (meaning that activation of CB2 or other SR144528-sensitive receptors accelerates emptying) or an indirect action (blockage of the receptor allows more molecules of WIN to be available to act on the CB1, delaying receptors), or both. Interestingly, prevention by AM251 of WIN-induced GI effects, particularly in the stomach, was complete only in combination with SR144528. Since, like most other cannabinoid antagonists, AM251 is not entirely specific to CB1 receptors, and at sufficient doses might also inactivate CB2 receptors,39 our results might indicate some AM251 binding to CB2 receptors, and some displacement from them by SR144528 when both drugs are given in combination. This is a provocative hypothesis warranting further investigation. Whatever the case may be, CB2 receptors (or the non-identified SR144528-sensitive site) could act as a brake for CB1 activation in the stomach.

As a consequence of gastric emptying being further delayed by administration of SR144528 prior to WIN, intestinal transit was also significantly delayed. Both mRNA for the CB2 receptor and the protein itself were found in different GI regions, including the small and large intestines.11 Furthermore, most myenteric neurones in the rat ileum showed immunoreactivity for this receptor.12 With the methodology applied here, the motility curves obtained for the small and large intestines are highly influenced by alterations in gastric emptying, and it can not be discarded, from our results, that the CB2 antagonist exerted a direct action on the intestine. However, this could be minor, since intestinal CB2 receptors are thought to be active only after an inflammatory stimulus.11,12,43–45

Finally, in contrast with AM251, no significant effect of the CB2 antagonists was found when they were administered alone, suggesting a lack of inverse agonist activity by SR144528 or AM630, or that endogenous cannabinoids tonically released under control conditions might not be as efficient upon CB2 as they are upon CB1 receptors.

Concluding remarks

Our results provide evidence, for the first time, that a site of action sensitive to the cannabinoid receptor antagonist SR144528 could modulate the effect of cannabinoids on GI motility in vivo. Further studies are needed to verify if gastric CB1 and CB2 (or other SR144528-sensitive) receptors exert opposing actions in regulating GI motility.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing Interests
  8. References

This work was supported by Ministerio de Educación y Ciencia (SAF2006-13391-C03-01; SAF2009-12422-C02-01), Universidad Rey Juan Carlos – Comunidad de Madrid (URJC-CM-2006-BIO-0604), and Comunidad de Madrid (S-SAL/0261/2006). SR144528 was kindly gifted by Sanofi-Aventis Recherche & Developpement (France). The authors wish to thank Óscar Gutiérrez (Servicio de Radiología, Clínica Universitaria, Universidad Rey Juan Carlos) for technical radiological advice.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing Interests
  8. References