Pharmacological Analysis of Cannabinoid Receptor Activity in Isolated Nerve-Smooth Muscle and Epithelial Preparations

This study was directed at characterising the cannabinoid receptor activity modulating the electrical field stimulation (EFS) evoked contractions of the rat isolated ileum myenteric plexus longitudinal muscle (MPLM) preparation, and the capsaicin, nicotine and veratridine evoked secretory responses of the rat isolated colonic submucosal plexus-mucosal (SPM) preparation. EFS of the MPLM preparation with single pulses at a repetition frequency of 0.05 Hz elicited a transient twitch contraction immediately in response to each electrical pulse. In contrast, stimulation of the MPLM preparation with 2 second trains of pulses every minute at a frequency of 30 Hz elicited a rapid transient rebound contraction on termination of each train of EFS. The non-selective cannabinoid receptor agonists AEA, CP 55,940, 9-THC and WIN 55,212-2 inhibited both EFS-evoked twitch and rebound contractions of the rat ileum MPLM elicited by 0.05 Hz and 30 Hz EFS respectively. The inhibition of the twitch contractions was competitively antagonised by the cannabinoid CB1 receptor antagonist / inverse agonist SR 141716 with pKB values of 8.60. In contrast, SR 141716 only antagonised the ability of AEA, 9-THC and WIN 55,212-2 but not CP 55,940 to inhibit the rebound contractions with pA2 values of 6.60. These observations extended to the inhibitory effect of WIN 55,212-2 on the twitch and rebound contractions of the guinea-pig ileum MPLM. The CB2 antagonist / inverse agonist SR 144528 did not alter the effects of the agonists. Additionally, the inhibitory effect of AEA was refractory to the vanilloid TRPV1 receptor antagonist capsazepine. WIN 55,212-3 a stereoisomer of WIN 55,212-2 was without effect on the rat MPLM. SR 141716 alone concentration-dependently increased the twitch contractions but inhibited the rebound contractions. Both types of the EFS-evoked contractions were abolished by the Na+ channel blocker tetrodotoxin or the muscarinic acetylcholine (ACh) receptor antagonist atropine but not the nicotinic ACh receptor antagonist hexamethonium. None of the cannabinoids altered the contractions to exogenously applied ACh. These data suggested that the cannabinoid agonists inhibited the twitch contractions through a stereospecific presynaptic CB1 receptor-mediated reduction in the release of ACh. Additionally, the inhibition of the rebound contractions occurred because of an inhibition of ACh release by a novel stereospecific presynaptic non-CB1 -non CB2 -non -TRPV1 site. The ability of SR 141716 to inhibit the rebound contractions and antagonise AEA, 9-THC and WIN 55,212-2 may be though partial agonism at the non-CB1-non CB2-non-TRPV1 site. The ability of SR 141716 to potentiate the twitch contractions by increasing the release of ACh suggested that the CB1 receptor was constitutively active or was subjected to a tonic activation by endocannabinoid agonists. A comparison between the maximal enhancement of the twitch contractions of the rat and the guinea-pig ileum MPLM caused by three CB1 receptor antagonists / inverse agonists AM 251, SR 141716 and O-2050 showed that each cannabinoid had a different maximum. This suggested inverse agonism. These data were supported with studies showing the lack of effect of three fatty acid amide hydrolase (FAAH) inhibitors AA-5HT, PMSF, URB–597 and VDM-11, an inhibitor of the AEA uptake transporter on EFS-evoked contractions. These studies showed that all three FAAH inhibitors increased the potency of exogenously applied AEA but not WIN 55,212-2, and that VDM-11 had no effect on the potency of exogenously applied AEA. This data suggested that a functional endocannabinoid tone and the uptake transporter were not present in the MPLM, but FAAH was present. These data provide supporting evidence that SR 141716 behaved as an inverse agonist in the MPLM to augment twitch contractions. The interaction between CP 55,940 or WIN 55,212-2 with SR 141716 was investigated using the rat colonic SPM sheet. Both CP 55,940 and WIN 55,212-2 attenuated the secretory responses to capsaicin and nicotine in a SR 141716 sensitive manner. SR 140333, a neurokinin 1 receptor antagonist, abolished the capsaicin and nicotine. This suggested that CP 55,940 and WIN 55,212-2 inhibited the capsaicin and nicotine response through a CB1 receptor-mediated inhibition of the release of substance P or neurokinin A. The sensitivity of the veratridine response to TTX and -chymotrypsin and the failure of the cannabinoids to attenuate the response suggested the absence of the CB1 receptor on the neurones releasing the undetermined neuropeptide. Together, these data suggest that both the CB1 receptor and non-CB1-non-CB2 -non-TRPV1 receptor can mediate the inhibitory effects of cannabinoid agonists in the rat ileum MPLM depending on the frequency of EFS. These data also show that SR 141716 is an inverse agonist in the MPLM. In the SPM preparation, the CB1 receptor appears to be involved in the modulation of some forms of peptidergic transmission