Effects on Excitatory Neuronal Pathways

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The depressant effects of cannabinoid receptor activation on gastrointestinal motility, as observed in vitro are, principally, the inhibition of evoked cholinergic and NANC contractile responses. Studies have focussed on the inhibition of the peristaltic reflex in segments of whole intestine, on the inhibition of evoked contractions of longitudinal or circular smooth muscle preparations or on the reduction of excitatory neurotransmitter release. Early experiments with 49-THC and some of the more non-polar organic fractions of tincture of Cannabis (British Pharmaceutical Codex) indicated the ability of putative cannabinoid receptor agonists to inhibit the contractile responses of the guinea-pig ileum without affecting responses to exogenous ACh (see review by Pertwee 2001). The peristaltic reflex can be reproduced in intestinal segments maintained in vitro. The synthetic cannabi-noid receptor agonists WIN 55,212-2 (0.3-300 nM) significantly decreased longitudinal muscle reflex contraction, compliance and maximal ejection pressure, while increasing the threshold pressure and volume required to elicit peristalsis in guinea-pigs (Izzo et al. 2000a). At maximal agonist concentrations, peristalsis was completely prevented. These effects were insensitive to the opioid antagonist naloxone, the a2-adrenoceptor antagonist, phentolamine or the CB2 receptor selective antagonist SR144528 (0.1 ^M). However, blockade was achieved with the CB1 receptor-selective antagonist SR141716A (0.1 ^M), thus indicating selective activation of cannabinoid CB1 receptors. Methanandamide, a more stable analogue of anandamide, similarly increased the peristaltic pressure threshold and inhibited the ascending circular muscle contraction (Heinemann et al. 1999). The methanandamide response was antagonised by SR141716A and also by apamin and reduced by the NO synthase inhibitor, N-nitro-L-arginine methyl ester (l-NAME) implying a possible involvement of apamin-sensitive Ca2+-activated K+ channels and nitric oxide (Heinemann et al. 1999). Thus, inhibition by cannabinoids may affect excitatory or inhibitory components of the reflex. These data are consistent with the ability of apamin to reduce cannabinoid CB1-mediated inhibition of cholinergic transmission in the guinea-pig ileum (Izzo et al. 1998).

Paton and Zar (1968) described the dissection of the MP-LMP of the guinea-pig small intestine. This preparation has been invaluable in the study of neurotransmission from the myenteric plexus to the longitudinal smooth muscle, particularly by opioids and cannabinoids, without the confounding effects of the peristaltic reflex. A similar preparation has been used to study neuromuscular transmission to the circular smooth muscle (Izzo et al. 1998). Contractions of MP-LMP induced by electrical field stimulation (EFS) were potently inhibited in a concentration-dependent fashion by the cannabinoid receptor agonists CP 55,940, CP 50,556, WIN 55,212-2, nabilone, CP 56,667,49-THC and cannabinol (Coutts and Pertwee 1997; Pertwee 2001). This inhibition was competitively and reversibly antagonised by SR141716A, without any effect on the inhibitory responses to normorphine

(^-opioid receptor agonist) or clonidine (a2-adrenoceptor agonist) and indicated an involvement of CB1 receptors. Therefore, electrically stimulated isolated preparations from the guinea-pig ileum have been used to demonstrate the high potency and stereoselectivity of CB1 receptor agonists (Nye et al. 1985; Pertwee 2001; Per-twee et al. 1992,1995,1996). The rank order of potency of agonists correlates well with their affinities for CB1 receptor binding sites in brain tissue and their known psychotropic effects (Pertwee 1997; Pertwee et al. 1992, 1996). The findings that the cannabinoid-induced inhibition of the guinea-pig MP-LMP was augmented by lowering the extracellular calcium concentration or attenuated by incubating the tissue with forskolin, 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP) or with the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine supports the known signal transduction mechanisms for CB1 receptors (Coutts and Per-twee 1998). Similar cannabinoid inhibitory effects on evoked responses have been reported for longitudinal strips of human tissue (Croci et al. 1998).

In a single electrophysiological analysis of intracellular recordings from myenteric neurons of the guinea-pig MP-LMP, WIN 55,212-2 or CP 55,940 were found to inhibit fast and slow excitatory synaptic transmission. In a subset of the neurons tested, this effect was reversed by SR141716A (Lopez-Redondo et al. 1997). Both cholinergic and NANC responses of circular smooth muscle due to EFS were presynaptically inhibited by cannabinoids by a mechanism that was sensitive to SR141716A but not l-NAME or naloxone (Izzo et al. 1998). Only the cholinergic component of this response was sensitive to attenuation by apamin, suggesting the involvement of Ca2+-activated K+ channels. The contractile responses to y-aminobutyric acid or 5-hydroxytryptamine, agents that release ACh in the intestine, have been shown to be reduced by49-THC or its analogues (Rosell and Agurell 1975; Rosell et al. 1976). There is some evidence that the release of adenosine, which also inhibits cholinergic neuromuscular transmission in this preparation, is susceptible to modulation via CB1 receptor activation (Begg et al. 2002a).

Effects on Inhibitory Neurotransmission

There is evidence that cannabinoids affect enteric inhibitory transmission in rodents. Storr and colleagues used standard intracellular recording techniques to study the effect of cannabinoid drugs on enteric transmission (Storr et al. 2004). Focal electrical stimulation of intrinsic neurons of isolated strips of the mouse proximal colon induced a transient excitatory junction potential (EJP, abolished by atropine) followed by a fast (transient) inhibitory junction potential (fIJP, which represents the apamin-sensitive component of inhibitory transmission) and a slow (sustained) inhibitory junction potential (sIJP, which represents the nitric oxide-dependent component of inhibitory transmission). WIN 55,212-2 significantly reduced EJP and the fIJP (an effect sensitive to the CB1 receptor antagonist SR141716A), but not sIJP; given alone, SR141716A significantly increased EJP, while fIJP and sIJP remained unchanged (Storr et al. 2004). These data suggest that cannabinoids, via CB1 receptor activation, might reduce the apamin component (which is mediated by ATP or related purines) of the inhibitory transmission in the mouse colon. Other indirect evidence was provided by Heinemann and colleagues, which showed that methanandamide depressed intestinal peristalsis with a mechanism involving, at least in part, facilitation of inhibitory pathways operating via apamin-sensitive K+ channels and nitric oxide (Heinemann et al. 1999) as mentioned above (Sect. 3.1.1). The effects of cannabinoids on the smooth muscle relaxation of the isolated gastric fundus in response to EFS of NANC innervation are not clear. In rat preparations (Storr et al. 2002), both excitatory cholinergic and NANC transmission were reduced by WIN 55,212-2 and anandamide. Only the anandamide responses were antagonised by the cannabinoid receptor antagonist AM630. By itself, AM630 had no effect on the contractile responses but facilitated the relaxation. This latter effect implied the presence of an ongoing endocannabi-noid tone that reduced the NANC neurotransmission. In contrast, Todorov et al. (2003) found no response to anandamide (0.1-10 ^M) in the isolated gastric fundus of the guinea-pig. Whether this is due to a species difference or whether the anan-damide was metabolised before it could produce a measurable response is unclear. No other, more potent cannabinoid receptor agonist was tested in this study, in which evidence suggested that the NANC response was mediated by nitric oxide and cyclic guanosine monophosphate (cGMP).

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