A CB1 ‘tone’ controlling intestinal motility: to be or not to be?
Anatomical and functional evidence suggests the presence of CB1 receptors in neurons of the myenteric plexus in a variety of species, including humans. Activation of prejunctional CB1 receptors reduces excitatory enteric transmission (mainly cholinergic transmission) in different regions of the GI tract, thereby leading to inhibition of motility (reviewed in7). There has been a debate as to whether, under physiological conditions, endocannabinoids tonically activate CB1 receptors to control small intestine and colon motility. Initial studies had suggested this possibility based on the observation that: (i) endocannabinoid levels in several districts of the GI tract are sufficient to constitutively activate CB1 receptors; (ii) CB1 antagonists increase motility, which parallels both their stimulation of electrically induced contractions of the guinea pig ileum in vitro and the finding of increased motility in CB1 receptor knockout mice; and (iii) blockade of endocannabinoid catabolism with selective inhibitors reduces intestinal and colonic motility.7 Importantly, as shown in a paper published in the Journal, CB1 antagonists as well as ‘knockout’ of CB1 also modulate other neurophysiological correlates of small intestine propulsion, such as the ascending neuronal contraction following electrical field stimulation of the rat ileum.8 The use of this set-up allowed the measurement of peristaltic activity and to separate the aboral stimulation site from the oral one, and led the authors to confirm that endocannabinoids and CB1 receptors are physiologically involved in the control of small intestine motility by inhibiting activity at the neuromuscular junction.8,9 Finally, an elegant in vitro study, published again in the Journal,10 showed how, in primary cultures of guinea pig myenteric neurons, CB1 receptor antagonists increase, and agonists decrease, spontaneous network activity as well as the number of: (i) synaptic vesicles being recycled during electrical stimulation; (ii) synaptophysin-immunopositive release sites; and (iii) mitochondria transported towards enteric fiber terminals, which are all specific indicators of prejunctional synaptic activity of myenteric neurons. The effects of the agonists could also be reproduced with two inhibitors of anandamide inactivation, thus again pointing to a constitutive control of myenteric neuron activity by the ECS.10
The conclusion from studies using cannabinoid antagonists that endocannabinoids exert tonic modulation of CB1 to inhibit motility was recently questioned on the basis that these compounds are not ‘neutral’ antagonists, but behave as inverse agonists in vitro at concentrations not too far from those corresponding to their Ki’s for CB1 receptors. This would suggest that the observed stimulation of motility by these compounds is not the result of their antagonizing the effects of endocannabinoid levels, but instead is due to their stabilization of a receptor conformation that has stronger affinity for the inactive form of the G-protein. It was argued that such possibility could be investigated using a new generation of ‘neutral’ CB1 antagonists now available,11,12 as these compounds would produce stimulation of GI transit only in the presence of endocannabinoid levels sufficiently high to activate CB1 receptors. Indeed, Storr et al., reported that one such compound, AM4113, unlike the widely used inverse agonist AM251, was devoid of any stimulatory activity on electrically induced contractions of the mouse ileum in vitro, although, somehow paradoxically, it did enhance upper intestinal transit, whereas it produced no stimulation of whole gut transit.11 The issue of ‘neutral’vs‘inverse agonism’ is tricky and very difficult to investigate in vivo, and a mathematical model has been proposed recently according to which all supposedly ‘neutral’ antagonists would exert inverse agonism in vitro provided that a sufficiently high, but still specific, concentration is used.13 Nevertheless, a somehow conservative interpretation of these results is that, while there might still be a tonic ECS controlling motility in the upper GI tract, further investigations are needed for the large intestine, although a study showed the depressant effect of an inhibitor of endocannabinoid inactivation on colonic propulsion.14
Pancreatitis, irritable bowel syndrome and septic ileus: is CB1 the ‘bad guy’?
Seminal studies carried out in the mid-2000s (reviewed in15) showed for the first time that the ECS, both in terms of endocannabinoid levels and CB1 receptor expression, is up-regulated with pro-homeostatic and protective function during several different types of experimental small intestine and colon inflammation, and that such up-regulation occurs also in human inflammatory bowel diseases (IBD). Recent reports (see below) have highlighted the role that CB2 receptors may also play in the taming of colonic inflammation and its consequences on motility, a possibility that, given the potential central side effects of CB1 receptor agonists, opens the way to the possible use of non-psychotropic CB2 agonists for the treatment of IBD, along with compounds that inhibit endocannabinoid inactivation. The anti-inflammatory effects of CB1/CB2 agonists have been recently studied also in the pancreas, as described in an article published in the Journal, through experiments carried out both in vitro, in isolated pancreatic acini, and in vivo, in experimental pancreatitis in rats.16 The authors showed that the cannabinoid receptor agonist, WIN55,212-2, inhibits the release of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) from acinar cells obtained from untreated rats, and reduced serum amylase, pancreatic edema and IL-6 and MCP-1 acinar content in rats with caerulein-induced pancreatitis, whilst also improving pancreatic damage in these animals. Interestingly, however, these protective effects were observed in vivo only when the CB1/CB2 agonist was given before the inflammatory stimulus, whereas when WIN55,212-2 was administered afterwards, the pancreatitis was worsened. While the protective effect observed with pretreatment was antagonized by a selective CB2 receptor blocker, the worsening effect was instead antagonized by a CB1-selective blocker.16 The authors suggested that, in the context of pancreatitis in vivo, CB1 activation might concur to oxidative stress or exert chemoattractant activity on macrophages, thus contributing to inflammation. An alternative explanation, however, might lie in the previous observation that, in the same experimental model of pancreatitis, TRPV1 channels participate in inflammation via a sensory mechanism leading to the production of pro-inflammatory peptides,17 and the same has also been reported for TRP channels of ankirin-1 type (TRPA1) in mice.18 The possibility exists that WIN55,212-2 might worsen pancreatitis through the sensitization of TRPV1 either via a direct interaction with this channel in a complex with TRPA1,19 or, indirectly, by activating CB1 receptors.20,21
In view of the very efficacious effects of endocannabinoid-based drugs in animal models of visceral pain, the role of the ECS in the control of irritable bowel syndrome (IBS) has also been proposed (reviewed in22). This hypothesis is supported by the recent finding of an association between a polymorphism in the Cnr1 gene encoding for CB1 receptors and the occurrence of IBS in the Korean population.23 Two studies published in the Journal have now addressed this possibility, using completely different approaches. Yüce et al., investigated the effect of CB1 agonists and antagonists/inverse agonists on afferent nerve discharges from rat myenteric neurons stimulated with either serotonin or bradykinin, two mediators known to activate sensory GI afferents and participate in visceral sensitivity.24 The results were intriguing and perhaps surprising in as much as the authors reported different effects of the agonist WIN55,212-2 (the activity of which on peristaltic activity was shown by the same group to be mostly mediated by CB1 receptors8) depending on the type of the stimulus, and possibly in a direction opposite to what expected. While WIN55,212-2 enhanced the effect of serotonin and did not influence that of bradykinin, the CB1 inverse agonist SR141716A (rimonabant) reduced the effect of bradykinin without affecting that of serotonin. Although counterintuitive, the findings with rimonabant might help to explain some anti-inflammatory effects observed in vivo with this compound in mice treated with lipopolysaccharide (LPS).25 The lack of effect of WIN55,212-2 on bradykinin, and its stimulation of the serotonin effect, instead, might argue against the possible therapeutic use of CB1 agonists in IBS, although of course studies in more specific animal models of this disorder should be carried out before reaching this conclusion. Interestingly, however, in the other study on this issue published very recently in the Journal, THC failed to produce any relief of visceral sensitivity after rectal distension in both healthy volunteers and IBS patients.26
Another GI disorder that might be ameliorated by antagonizing, rather than enhancing, the activity of CB1 is ileus, a pathological state consisting of decreased intestinal motility following peritonitis, surgery, or other noxious situations. Mascolo et al., showed that, in acetic acid-induced ileus in mice, reduced intestinal motility was accompanied by increased levels of anandamide compared with control mice, and by overexpression of CB1 receptors in myenteric nerves.27 Importantly, reduced transit was alleviated by rimonabant, but not by a CB2-selective antagonist, and was worsened by VDM11, a selective inhibitor of anandamide cellular uptake.27 In an article published in the Journal, Li et al., show that not only CB1, but also CB2 receptors might participate in LPS-induced ileus in rats, a model of septic ileus.28 In this case, the authors monitored not only upper intestinal motility but also spontaneous jejunal myoelectrical activity and IL-6 and tumor necrosis factor (TNF)-α release, and found that antagonism not only of CB1, but also of CB2 receptors, prevented LPS-induced reduction of myoelectrical activity and of upper GI transit. CB1 and CB2 antagonists also tended to reduce the elevation of IL-6 induced by a low dose of LPS.28 These data indicate that, contrary to ileus induced by a chemical irritant, also CB2 receptors participate in the etiopathology of septic ileus, possibly because of their role in inflammation. Furthermore, they also confirm the role of CB2 receptors in regulating intestinal motility under inflammatory conditions (or perhaps not just [see below]?).
Although initially neglected, the study of the role of the ECS in the control of gastric motility has been recently investigated in several studies, two of which published in the Journal. The existence of a CB1 tone controlling gastric emptying was first suggested by data indicating that: (i) anandamide inhibits this function in a way counteracted by the CB1 receptor antagonist rimonabant, but not by the CB2 receptor antagonist SR144528 or by TRPV1 antagonist 5′-iodoresiniferatoxin; (ii) inhibition of anandamide degradation by fatty acid amide hydrolase (FAAH) also reduces gastric emptying in a way partly reduced by rimonabant; and (iii) rimonabant per se increases gastric motility.29 Interestingly, the inhibitory effect on gastric transit by CB1 activation, as recently investigated by the use of WIN55,212-2 and the CB1 antagonst AM251, does not undergo tolerance following chronic stimulation, unlike the inhibition of upper intestinal or colorectal transit, or the psychotropic effects of chronic CB1 agonism.30 This finding should open the way to future mechanistic studies investigating the molecular bases of this lack of tolerance, which might be due, for example, to impaired CB1 receptor internalization following repeated stimulation in the stomach. Furthermore, since delayed gastric transit may contribute to satiety and emesis, the authors suggested that the lack of tolerance to inhibition of gastric motility following chronic administration with CB1 agonists might reduce the efficacy of these compounds as anti-anorexiant and anti-emetic therapies.31 Nevertheless, WIN55,212-2 was recently shown to inhibit gastric myoelectric function, in terms of reduction of the frequency of antral pacemaker activity, both in vehicle- and apomorphine-treated ferrets.31 Although no CB1 antagonist was used in this study to ascertain the involvement of CB1 receptors in the effects of the compound, these data provided further substantiation to the well-known anti-emetic actions of CB1 receptor activation (reviewed in32), and in fact WIN55,212-2 was found by the authors to inhibit also the apomorphine-induced emetic response.31 On the other hand, contrary to previous findings obtained in the ferret using a different pro-emetic stimulus,33 the authors found that the FAAH inhibitor URB597 did not reduce retches and vomits induced by the non-selective dopamine receptor agonist.31