Elsevier

Pharmacological Research

Volume 56, Issue 2, August 2007, Pages 132-139
Pharmacological Research

Evidence for a modulatory role of cannabinoids on the excitatory NANC neurotransmission in mouse colon

https://doi.org/10.1016/j.phrs.2007.04.019Get rights and content

Abstract

It is well accepted that endogenous cannabinoids and CB1 receptors are involved in the regulation of smooth muscle contractility and intestinal motility, through a mechanism mainly related to reduction of acetylcholine release from cholinergic nerve endings. Because, few data exist on a possible modulatory action of the cannabinoid agents on the non-adrenergic non-cholinergic (NANC) excitatory and inhibitory neurotransmission, the aim of the present study was to investigate the effects of cannabinoid drugs on the NANC responses elicited by electrical field stimulation (EFS) in the circular muscle of mouse proximal colon. Colonic contractions were monitored as changes in endoluminal pressure.

In NANC conditions, EFS evoked TTX-sensitive responses, characterized by a relaxation, nitrergic in origin, followed by a contraction. The EFS-evoked contraction was significantly reduced by SR48968, NK2 receptor antagonist, and abolished by co-administration of SR48968 and SR140333, NK1 receptor antagonist, suggesting that it was due to release of tachykinins. The cannabinoid receptor synthetic agonist, WIN55,212-2, the putative endogenous ligand, anandamide, the selective CB1 receptor agonist ACEA, but not the selective CB2 receptor agonist JWH-015, produced a concentration-dependent reduction of the NANC contractile responses, without affecting the NANC relaxation. ACEA or anandamide did not modify the contractions induced by exogenous [β-Ala8]-NKA(4–10), agonist of NK2 receptors. The selective antagonist of CB1 receptors, SR141716A, per se failed to affect the EFS-evoked responses, but antagonized the inhibitory effects of WIN55,212-2, anandamide and ACEA on NANC contractile responses. AM630, CB2 receptor antagonist, did not modify the inhibitory effects of WIN55,212-2 or anandamide. URB597, inhibitor of the fatty acid amide hydrolase, enzyme which catalyze the hydrolysis of anandamide, was without any effect on the NANC evoked responses. We conclude that the activation of prejunctional CB1 receptors produces inhibition of NANC contractile responses in mouse colonic preparations. However, endogenous ligands do not seem to modulate tonically the NANC transmission in mouse colon.

Introduction

Cannabinoids have been shown to exert a broad variety of pharmacological actions, including central and peripheral effects, through receptor-mediated mechanisms [1]. Many of the reported pharmacological effects are mediated by two specific receptors, denoted CB1 and CB2 receptors, both coupled to G-proteins. CB1 receptors are found mainly in central and peripheral neurons, while CB2 receptors are located in peripheral tissues, particularly in immune cells [2]. Endogenous ligands for these receptors include arachidonylethanolamide (anandamide), 2-arachidonylglycerol (2-AG) and noladin ether. Inactivation of endocannabinoid signalling is dependent on cellular uptake, intracellular transport and enzymatic hydrolysis by the fatty acid amide hydrolase (FAAH) [3]. Therefore, the “endocannabinoid system” comprises the cannabinoid receptors, their endogenous ligands and the proteins participating in the inactivation of these latter compounds [4].

In the gastrointestinal tract CB1 receptors appear to be involved in several physiological process, including gastric secretion, intestinal motility, gastrointestinal transit and colonic propulsion [5], [6], [7], [8]. In agreement with these observations, CB1 are present in the gastrointestinal tract and are localized in neurons of the myenteric and submucosal plexuses in a variety of species, co-expressed with cholinergic markers [9], [10], [11], [12], [13]. Endogenous ligands, anandamide and 2-AG have been detected also in the mouse small intestine [14]. In the mouse gastrointestinal tract the highest levels of CB1 receptors have been detected in stomach and colon and CB1 immunoreactivity is present in ganglia and in the smooth muscle layers of both the small and large intestine [15].

It is well accepted that the mechanism by which the activation of CB1 regulates smooth muscle contractility and intestinal motility is related to reduction of acetylcholine release from cholinergic nerve endings [16], [17]. However, other mechanisms, such as activation of vanilloid receptor [18], [19], modulation of adenosine release [20], stimulation of myenteric cholinergic neurons through lipoxygenase metabolites [21] have been also proposed.

In the last decade, non-adrenergic non-cholinergic (NANC) excitatory transmission has been object of vigorous research because it has become clear that it contributes to the intestinal motor function and its role becomes even more important in a pathophysiological setting. In particular, tachykinins, acting as co-transmitters in the neuromuscular transmission, function as a backup system during peristalsis and a reduction in the ability of tachykinin receptor agonists to contract the intestinal smooth muscle has been reported in various disorders of the gastrointestinal tract [22]. Inhibition of NANC excitatory responses by cannabinoid agonists has been reported just in guinea-pig circular muscle [17]. Moreover, few studies have investigated the effects of CB ligands on the intestinal NANC inhibitory neurotransmission. However, those that have appear not to be concordant, because facilitation in guinea-pig ileum [23], inhibition in rat gastric fundus [24] or no effect in human colon [25] have been reported.

Therefore, the purpose of the present study was to verify whether cannabinoid drugs could influence the NANC excitatory and inhibitory neural transmission in mouse proximal colon. For this aim, we used the exogenous and endogenous non-selective CB receptor agonists, respectively, WIN 55,212-2 and anandamide; the selective CB1 agonist ACEA; the selective CB2 agonist JWH-015; the selective CB1 and CB2 antagonists, respectively SR141716A and AM630. Data from the study were presented at the 1st Joint International Society Meeting in Neurogastroenterology and Motility [26].

Section snippets

Materials and methods

Experimental procedures were in accordance with the European Communities Council Directive of 24 November 1986 and were approved by Ministero della Sanità (Rome, Italy). Adult mice (C57BL/10) were killed by cervical dislocation. The abdomen was immediately opened and the proximal colon was removed distally to caecum. The colonic lumen was cleaned with Krebs solution and segments of about 2 cm in length were cut. Preparations were mounted in a custom designed horizontal organ bath, which was

NANC responses induced by EFS

In mouse colonic preparations, EFS (2–32 Hz) evoked consistent responses, which were characterized by an early transient relaxation followed by a contraction. The responses were abolished by TTX (100 nM, n = 3), indicating their neural origin. The relaxation was abolished by l-NAME (300 μM), inhibitor of nitric oxide (NO) synthesis, indicating that it was nitrergic in origin. As previously described [28], the EFS-evoked contractions were markedly reduced by SR48968 (0.1 μM), a selective NK2 receptor

Discussion

The results of the present study suggest that activation of prejunctional cannabinoid CB1 receptors is able to modulate the NANC excitatory transmission in mouse colonic preparations, without affecting the inhibitory transmission. In addition, the NANC evoked contractions do not appear to be under a tonic influence by endogenous cannabinoids.

Several immunohistochemical and pharmacological studies have shown the role of CB1 receptors in the inhibition of motility in the mouse small and large

Acknowledgements

This work was supported by a grant from Ministero dell’Università e della Ricerca scientifica (MIUR), Italy. We thank Sanofi Recherche (Montpellier Cédex, France) for supplying SR141716A, SR140333 and SR48968.

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