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.