In the present study, we have sought to determine the effects of the CB1 receptor agonist, ACEA, on enteric neuronal and colonic longitudinal and CM function in humans. In addition, immunohistochemical studies examined the distribution of the CB1 receptor in the human colon in relation to a marker of cholinergic neurons, ChAT.
Properties of neurogenic contractility in isolated human colonic muscle strips
EFS of human colonic muscle strips in the presence of an NOS inhibitor evoked two distinct types of contractions, an ‘on-contraction’ during the period of electrical stimulation and an ‘off-contraction’ initiated after the stimulus had ceased. This pattern of neurogenic contractility in response to EFS has been previously described in isolated human colonic tissue (Mitolo-Chieppa et al., 1998; 2001; Tomita et al., 1998; McKirdy et al., 2004), where the off-contraction has been likened to the equivalent of the oral ascending contractile component of peristalsis in the isolated tissue preparation (Mitolo-Chieppa et al., 2001). The experimental conditions partly determine the extent of on-contraction, as EFS would normally concomitantly activate both excitatory cholinergic and inhibitory nitrergic transmission in vitro, producing a degree of physiological antagonism of both types of contraction (McKirdy et al., 2004). Evidence suggests that both types of contractions are neurogenic in origin, as both are abolished by tetrodotoxin (Tomita et al., 1998).
Effects of CB1 receptor agonists on excitatory motor responses
The CB1 receptor agonist ACEA inhibited the on-contractions of human colonic circular, but not longitudinal, muscle preparations. The relative lack of CB1 immunolabelling in the LM layer, combined with a lack of functional effect of ACEA on longitudinal neurogenic contractility, suggests that CB1 receptors modulate motility primarily through an action within the CM layer of the human colon. The innervation and regulation of LM is more variable and less well understood than that of CM, but studies do suggest a high proportion of cholinergic motorneurones in LM layers (Bornstein et al., 2004). The findings of the present study suggest that functional CB1 receptors are localised primarily to circularly projecting cholinergic motorneurones in the human colon, which is at variance with previous studies (Manara et al., 2002). The reasons for such differences are not clear, but functionally may relate to differences in experimental conditions, including the use of different CB1 receptor agonists and other pharmacological pretreatments, especially with the use of indomethacin in other studies. Indomethacin has been shown to directly inhibit fatty acid amide hydrolase, the enzyme responsible for the degradation of endogenous CBs such as anandamide (Fowler et al., 2003). As such endocannabinoids are produced and exert functional inhibition of motility during EFS in isolated gastrointestinal preparations (Izzo et al., 1998), there may be a potentiation of CB effects from EFS in the presence of such a pretreatment. Indomethacin has also been demonstrated to potentiate neurogenic excitatory LM contractions in human colon through both pre-and postjunctional sites via cyclooxygenase (COX) inhibition (Fornai et al., 2005). This may have also contributed to the facilitation of, or otherwise revealed an effect of, CBs in longitudinal preparations ‘sensitised’ to contraction. Indeed, the differential expression of COX isoforms between circular and LM layers (Fornai et al., 2005) infers a potentially complex role for prostanoids in the control of gastrointestinal motility and the use of indomethacin needs to be considered judiciously in this setting.
The inhibitory effect of ACEA on EFS-evoked contractions was reversed when ACEA was incubated in the presence of the CB1 receptor-selective antagonist AM251. This finding suggests that the inhibitory action of ACEA was being achieved through selective activation of CB1 receptors and is in keeping with previous studies which have demonstrated a reversal of CB agonist-evoked inhibition of neurogenic cholinergic contractility following pretreament with a CB1-receptor antagonist (Coutts & Pertwee, 1997; Croci et al., 1998b; Izzo et al., 1998; Manara et al., 2002).
ACEA inhibited neither the maximal contraction of ACh nor the NK-2 receptor-selective agonist, β-ala8-NKA. Similarly, the potency of ACh in evoking 50% of the maximal contraction was unaffected by ACEA in either LM or CM. As both agents evoke contraction primarily by activating receptors directly on the smooth muscle (Croci et al., 1998a, 1998b), the results indicate that the inhibitory action of ACEA on cholinergic transmission is achieved primarily by acting at prejunctional or presynaptic CB1 receptors. These findings are consistent with previous studies which have described the prejunctional locus of the inhibitory effect of CBs on neurogenic ACh release from a variety of visceral preparations (Coutts & Pertwee, 1997; 1998; Croci et al., 1998b; Izzo et al., 1998; Spicuzza et al., 2000). In addition, our immunohistochemical studies support a neuronal site of location of the CB1 receptor.
Effects of CB1 receptor agonists on inhibitory (relaxation) motor responses
Following precontraction and under NANC conditions, EFS caused frequency-dependent relaxation of both circular and LM preparations. Previous studies have demonstrated that the EFS-evoked NANC relaxation is mediated primarily by nitric oxide (Tomita et al., 1998; Zyromski et al., 2001) with possible corelease of ATP, vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide (Keef et al., 1993; Bornstein et al., 2004). Evidence of a small but nonsignificant enhancement of EFS-evoked relaxation in the presence of ACEA may be a permissive effect due to inhibition of a residual or atropine-resistant component of stimulated release of ACh, a neurokinin or serotonin. Alternatively, CB1 receptor activation may facilitate inhibitory motor pathways in the colon, leading to a more pronounced relaxation response. This has been demonstrated previously using methanandamide in the isolated guinea-pig ileum (Heinemann et al., 1999). A direct myogenic facilitation of relaxation cannot be excluded, but is unlikely, as ACEA did not evoke direct relaxation of human colonic tissue and isoprenaline-evoked relaxation was unaffected by ACEA pre-treatment (data not shown).
Immunohistochemical localisation of the CB1 receptor and colocalisation with ChAT
CB1-IR was distributed in nerve cell bodies and nerve fibres in select regions of the myenteric plexus, submucosa and in a number of distinct structures in the muscle layers. These findings are consistent with the reported distribution of CB1-IR in the porcine (Kulkarni-Narla & Brown, 2000), mouse (Pinto et al., 2002; Casu et al., 2003), rat and guinea-pig colon (Coutts et al., 2002). These data are also supported by the recent immunohistochemical localisation of CB1-IR in human colon (Wright et al., 2005), although the weak signal localised to circular and LM in that study was not demonstrated in the present study and it should be noted that many functional studies do not support a role for CB1 receptors acting postjunctionally on smooth muscle.
The distribution of CB1-IR was limited compared to the broad distribution of the cholinergic marker, ChAT. Immunoreactivity towards ChAT was detected broadly in the human colon, particularly in the myenteric plexus and submucosa. ChAT-positive nerve fibres occurred sporadically throughout the entire thickness of the submucosa, although these fibres were not arranged in any distinct nerve plexus. ChAT immunoreactive nerve fibres were also identified in the myenteric plexus and these fibres frequently extended into the circular and LM layers. ChAT-positive nerve fibres also coursed in thick concentric bands between the septa of the CM, with varicosities identified in both muscle layers but particularly in the CM. This pattern of distribution is similar to previous studies using human colon tissue (Porter et al., 1997; 2002; Schneider et al., 2001). Cells in the mucosa that demonstrated intense immunoreactivity towards ChAT are most likely to represent enteroendocrine cells (Porter et al., 1996).
Double-labelling studies examining the colocalisation of the CB1 receptor and ChAT revealed that CB1-IR was highly colocalised with immunoreactivity for ChAT in the myenteric plexus, submucosa and nerve fibres, extending predominantly into the circular and LM layers. Distinct neural populations that were immunoreactive for either ChAT or the CB1 receptor alone were also identified. It is likely that, in these neural populations, the CB1 receptor and ChAT were colocalised with other neurotransmitters such as NOS, VIP or substance P (Kulkarni-Narla et al., 1999; Kulkarni-Narla & Brown, 2000), reflecting the plurichemical nature of neurotransmission in the human colon (Porter et al., 1997).
CB1-IR was occasionally found in distinct, yet unidentified, structures in the CM layer. The fine mesh-like webbing was interspersed with densely labelled puncta in close proximity to a tract of dense labelling that did not colocalise with ChAT. It would be provocative to suggest that the morphology may be consistent with a sensory structure such as an intramuscular array, one of two types of terminal specialisations of extrinsic primary afferent neurones present in the gastrointestinal tract (Phillips & Powley, 2000). However, the structures identified in the present study are not dissimilar to such sensory terminals identified in the rat colon (Wang & Powley, 2000). Intrinsic primary afferent neurons also cannot be excluded, as nerves with Dogiel Type II morphology have been shown to project to CM in the human colon (Wattchow et al., 1997). Future studies utilising anterograde labelling of extrinsic nerves together with immunohistochemistry may discern the nature and chemical coding of these structures.
The capacity for CB1 receptor activation to reduce neurogenic contractility in the human colon provides support for the development of CBs as therapeutic agents in hypermotility disorders. However, such therapies may also be of value in the treatment of a wide spectrum of GI disorders such as irritable bowel syndrome (IBS), diarrhoea, diverticulosis and gastroesophageal reflux disease (Holzer, 2001; Hunt & Tougas, 2002; Di Carlo & Izzo, 2003). The localisation of the CB1 receptor in mucosal and submucosal neurones, as also described in the human colon in a recent study (Wright et al., 2005), may suggest a role in the modulation of mucosal secretory function. The CB1 receptor agonist WIN 55,212-2 has been demonstrated to reduce electrically evoked secretory responses in the rat and guinea-pig ileum by acting directly at CB1 receptors on enteric nerves (Tyler et al., 2000; MacNaughton et al., 2004).
CBs exhibit antiemetic, orexigenic and analgesic effects in addition to purportedly suppressing the development of colorectal malignancy (Ligresti et al., 2003). CB1 receptor upregulation in inflammatory conditions (Izzo et al., 2001a; Siegling et al., 2001) and the recent finding that CBs ameliorate the development of colonic inflammation (Massa et al., 2004) implicate CBs as modulators of the neuroimmune axis in the gastrointestinal tract (Gongora et al., 2004; Kraft et al., 2004). The recent finding that a CB2 receptor agonist was capable of reducing the enhanced gastrointestinal transit following an inflammatory stimulus (Mathison et al., 2004) suggests that further studies examining the role of both CB1 and CB2 receptors in human gastrointestinal disease are necessary in order to reveal the true therapeutic value of CBs.
In conclusion, this study has demonstrated that CB1 receptor activation inhibits the neurogenic contraction to EFS in the human colon; this effect is attributed to inhibition of cholinergic motorneuronal ACh release. This finding is supported by immunohistochemical studies revealing a high level of colocalisation between the CB1 receptor and ChAT in enteric neurones of the human colon.