In the present study, the acute and chronic effects of cannabinoid agonist administration in three different systems (central nervous, CV and GI) were analyzed in parallel in the rat. Under similar experimental conditions, the different systems show differential sensitivity to the effects of the mixed cannabinoid agonist WIN and the development of tolerance. Thus, although no alteration in CV parameters was detected after acute administration of WIN, it induced the typical central signs (cannabinoid tetrad), as well as depression of GI motility. After chronic administration, no CV effects, hypolocomotion, or hypothermia were detected, whereas delayed gastric emptying, analgesia and some catalepsy were still present, suggesting that these effects are relatively resistant to the development of tolerance.
Effects of acute administration of WIN
In the early 1970’s, several authors reported that THC reduced defecation in rats38,39 and delayed passage of a charcoal meal in mice.40 However, this is the first time that the effect of a cannabinoid on GI motility has been analyzed in experimental animals using radiographic techniques, which allow the analysis of different regions of the GI tract at several time points in the same individual.33
In agreement with previous reports,13,15,16,18,21 when acutely administered, the cannabinoid agonist WIN exerted its depressive action in both stomach and intestine, and this effect was transient.30 Thus, the stomach of high WIN-treated animals was practically inactive for 2 h, but afterwards, gastric emptying returned to normal, and the slope of the curve was similar to that for control animals (Fig. 2A).
Although the reduction in small intestinal transit described here may be influenced by delayed gastric emptying, the fact that low cannabinoid doses were effective in reducing intestinal transit and not gastric emptying supports the notion that cannabinoids depress intestinal motility independently of their effects on gastric emptying.13,18 Moreover, if WIN did not exert an independent action on the intestine, the radiographic motility curves for the small intestine, caecum and colorectum would have simply been displaced to the right but would have kept approximately the same shape/slope as in controls, as after treatment with other drugs that mainly delay gastric emptying.33 Instead, in rats treated with high-WIN, the curve for the small intestine was wider (Fig. 3A), and the slopes of those for caecum (Fig. 4A) and colorectum (Fig. 5A) were slower than in saline- or vehicle-treated rats. In addition, although our method does not allow for a quantitative analysis of peristaltic activity, peristaltic waves in the small intestine were less apparent after WIN administration (compare Fig. 3D,F with Fig. 6F), further supporting the existence of a direct action in the small intestine.
Our results also indicate that the intestine might be more sensitive than the stomach to the depressive effect of cannabinoids, which is in disagreement with other studies reporting similar sensitivity for both regions.13,15,16,18 In those studies, analyses were made within 1 h after drug administration using invasive techniques. In our study, alterations in motility curves of all GI regions were best detected 2–8 h after drug administration, but 1 h after high-WIN, gastric emptying tended to be slower, and motility in the small intestine was already significantly uncoupled.
Central and CV acute effects of WIN were evaluated at least 20 min and no later than 2 h after drug administration, while gastric emptying was still uncoupled. As expected, WIN induced the typical central effects.4,5,36 On the other hand, even though most cannabinoids induce both bradycardic and hypotensive effects in the rat,8,41 we did not detect any remaining effect 1 h after WIN administration. Differences in the time point chosen for recording and/or the methodology used might explain the discrepancy with other reports. In fact, we have observed that intraperitoneal WIN administration provokes a transient decrease in MBP (about 20 mmHg) and HR (about 30 beats min−1) in the rat, starting 5 min and lasting for 30–40 min after administration (unpublished observations).
Interestingly, these results suggest that different systems respond differently to the same cannabinoid treatment. In both GI tract and CNS, whose functions were acutely depressed by WIN, cannabinoids are known to exert their actions through a similar mechanism, the presynaptic inhibition of neurotransmitter release.17,42 In contrast, in the CV system, cannabinoids act on both nerve fibres innervating the tissue and the tissue itself.11
Effects of chronic administration of WIN
No alterations in MBP or HR were found after chronic administration of WIN. In contrast, the last administration of WIN still induced some effects on GI motility and CNS, whereas other cannabinoid-induced alterations were less intense or even disappeared.
In agreement with previous reports,30,43–46 some central effects were more resistant to attenuation after chronic treatment than others. Thus, tolerance was complete to hypothermia and hypolocomotion and partial to catalepsy, whereas antinociception remained unchanged, which might be a useful feature if cannabinoid agonists were used therapeutically.
Most studies of GI signs of tolerance have been carried out in vitro.17,31,32 Nevertheless, an early report showed that mice pretreated with THC at 10 mg kg−1 orally once daily for 2–4 days developed long-lasting tolerance to the inhibitory effect of this cannabinoid on the passage of a charcoal meal.30 In our study, tolerance to reduced intestinal transit (measured as arrival and filling of the caecum) developed after daily treatment for 14 days, but tolerance to gastric emptying delay did not develop to the same extent, and 4–8 h after the last high-WIN administration, a significant amount of barium was still present in the stomach. In agreement with previous reports testing WIN and other cannabinoid agonists,47–49 daily food intake and body weight gain were significantly lower in rats receiving high-WIN each day. Although anxiogenic effects50 and some depressed locomotor activity could have contributed, the fact that each administration delayed gastric emptying for about 2 h might have also influenced feeding and body weight gain.
It is likely that treatments longer than 14 days are needed to fully attenuate WIN-induced central and GI effects. In any case, our results support the idea that development of tolerance may be dependent on the particular cannabinoid effect and the anatomical region acted upon.46 Furthermore, it might involve different mechanisms for hypothermia, catalepsy, and reduction of intestinal transit on one hand, and for antinociception and gastric emptying delay, on the other.
As reported for other WIN chronic effects, like anxiogenic responses,50 1 week after the last dose, no residual GI or central effects were apparent. This is not surprising as the half life of WIN is 24–36 h.47,50 Thus, those cannabinoid effects that are resistant to tolerance are also relatively short-lasting. As acute or daily WIN administration did not produce any permanent MBP or HR changes, it seems that chronic administration of cannabinoids, even at high doses, might be relatively safe.
CB1 receptor involvement in GI motor function
Although cannabinoids may modulate GI function through central receptors,21,51 they are known to primarily decrease GI contractility and peristalsis by a direct action on the myenteric neurons.23,52–55 CB1 receptors, located in these neurons,25,26,56 are responsible for the effects on GI motility in vitro and in vivo.14,16 Although we have not totally excluded the involvement of CB2 receptors, which are now known to exist in the brain and, perhaps, in the peripheral nervous system, and have been implicated in emesis57 and changes in motility induced by lipopolysaccharide,58 the remaining GI effect (namely, gastric emptying delay) after daily WIN treatment was completely blocked by the CB1 antagonist AM 251, further confirming that CB1 receptors are important modulators of GI motor function, even after chronic cannabinoid treatment.
The CB1 antagonist showed some accelerating effect in most GI regions when administered alone, which is in agreement with previous reports suggesting that CB1 antagonists, including AM 251,59 might exert an inverse agonist action or unmask an endogenous cannabinoid inhibitory tone, in the GI tract.14,21,23,52,60,61 However, whereas in previous reports in which invasive methods were used, the CB1 antagonist rimonabant did not alter gastric emptying per se in fasted rats,15,18 we did find a slight transient accelerating effect on this parameter. Differences in methodology and/or the drug used might account for this discrepancy.
The therapeutic potential of chronic cannabinoid administration might be counteracted by the presence of persistent adverse effects. Our data indicate that CV function might remain well preserved but that some central and GI signs might still occur during chronic treatment. Thus, under similar experimental conditions, the different systems show differential sensitivity to the effects of cannabinoids and to the development of tolerance. Although chronic cannabinoid treatment, even at high doses, might be relatively safe, delayed gastric emptying seems particularly resistant to attenuation, and this could have some influence on feeding and weight gain. Therefore, our data suggest that monitoring GI motor function could improve safety of chronic cannabinoid treatment.