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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 18, 2002 as doi:10.1096/fj.02-0338fje. |
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Department of Experimental Pharmacology, University of Naples ‘Federico II’, 80131 Naples, Italy;
* Institute of Biomolecular Chemistry, National Research Council, Comprensorio Olivetti, Ed. 70, 80078 Pozzuoli (NA), Italy;
Department of Biological Structures, Functions and Technology, University of Naples ‘Federico II’, 80137 Naples, Italy; and
Department of Pharmaceutical Sciences, 84084 Fisciano (SA), Italy
2Correspondence: A.A.I., Department of Experimental Pharmacology, University of Naples ‘Federico II’, via D. Montesano 49, 80131 Naples, Italy. E-mail: aaizzo{at}unina.it; V.D.M., Institute of Biomolecular Chemistry, National Research Council, Via Campi Flegrei 34, Comprensorio Olivetti, Ed. 70, 80078 Pozzuoli (NA), Italy. E-mail: vdimarzo{at}icmib.na.cnr.it
SPECIFIC AIMS
Paralytic ileus is defined as a long-lasting inhibition of gastrointestinal motility in response to nociception initiated at the abdominal level. The molecular bases of this disease are not known. In this study, based on the inhibitory effects of endocannabinoids on intestinal motility and on the finding of enhanced endocannabinoid levels during nociception and tissue damage, we investigated the possibility that an enhanced tone of the endocannabinoid system is partly responsible for paralytic ileus in a mouse model of this disorder.
PRINCIPAL FINDINGS
1. Anandamide levels in the mouse small intestine are enhanced during ileus
Intraperitoneal administration of acetic acid strongly reduced gastrointestinal transit. A mild infiltration of leukocytes was observed in sections of intestine obtained from mice subjected to peritonitis compared with the sections of intestine of control mice. Table 1
shows that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) and the anandamide congener palmitoylethanolamide were detected in control tissues and that only levels of anandamide were significantly increased in the small intestine of acetic acid-treated mice.
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2. The density of cannabinoid CB1 receptors in enteric neurons is enhanced during ileus
In saline-treated animals, CB1 receptor immunoreactivity (CB1-IR) was observed in neural perikarya and fibers of the myenteric plexus, in nerve fibers running through the circular muscle layer (Fig. 1
), and rarely in nerve fibers through the submucosa and mucosa. Double labeling showed CB1-IR in ChAT-immunoreactive (ChAT-IR) myenteric perikarya, a marker of cholinergic neurons (Fig. 1b, b', c, c'
), and in fibers to the circular muscle (Fig. 1c, c'
). A few CB1-IR fibers that were not cholinergic as well as cholinergic nerve fibers that did not exhibit CB1-IR were observed (Fig. 1c, c'
]). CB1-IR was exhibited by substance P-IR and non-IR perikarya of the myenteric plexus (Fig. 1d, d'
). In acetic acid-treated animals, increased neural density of CB1-IR was observed within the interganglionic strands and ganglia of the myenteric plexus (Fig. 1e, f, h
), in nerve fibers to the circular muscle (Fig. 1e, h
), in submucosal neural perikarya and fibers (Fig. 1g
), and in nerve fibers that extended up the mucosa, among the glands (Fig. 1i
). CB1-IR was observed in myenteric (Fig. 1f, f'
) and submucosal ChAT-reactive perikarya (Fig. 1g, g'
), in substance P-IR perikarya and nerve fibers contained within the myenteric plexus (Fig. 1h, h', i, i'
), as well as in a part of substance P-containing nerve bundles and fibers to the circular muscle layer (Fig. 1h, h'
). The CB1-containing periglandular nerve fibers did not exhibit substance P-IR (Fig. 1i, i'
).
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3. The CB1 receptor antagonist SR141716A enhances gastrointestinal transit during ileus less potently than in control mice
The cannabinoid CB1 receptor antagonist SR141716A (1–20 mg/kg, i.p.), but not the CB2 antagonist SR144528 (1–10 mg/kg, i.p.), dose-dependently increased intestinal motility in mice with peritoneal irritation (P<0.01 and 0.001 with 10 and 20 mg/kg, respectively, one-way ANOVA followed by Tukey-Kramer multiple comparisons test). However, SR141716A was significantly more potent in control mice (ED50 0.406±0.07 mg/kg) than in acetic acid-treated mice (ED50 2.59±0.29 mg/kg). The effect of SR141716A (10 mg/kg, i.p.) was reduced by a per se ineffective dose of the selective CB1 receptor agonist, arachidonoyl-chloro-ethanolamide (ACEA, 0.3 mg/kg, i.p., P<0.01), but not by the selective CB2 receptor agonist JWH-015 (10 mg/kg, i.p., P>0.05). Higher doses of ACEA administered alone (1 mg/kg, i.p.) further delayed the impaired motility in acetic acid-treated mice (% transit: ileus 26±3; ileus+ACEA 16±2, n=12, P<0.05), whereas JWH-015 (10 mg/kg, i.p.) given alone was without effect (% transit: ileus 26±3; ileus+JWH-015 27±4; n=12, P>0.2). ACEA (0.3 mg/kg, i.p.) was unable to modify the prokinetic effect of carbachol (0.03 mg/kg, i.p.) in acid acetic-treated mice (% transit: ileus 24±4; ileus+carbachol 46±4; ileus+carbachol+ACEA 42±4, n=12). Saline, DMSO, and ethanol (4 µL/mouse, i.c.v. or 10 µL/mouse, i.p.) had no effect on the response under study either in control or in acetic acid-treated mice.
4. The anandamide transporter inhibitor VDM11 worsens intestinal hypomotility during ileus
The selective inhibitor of the anandamide membrane transporter, VDM11 (10 mg/kg, i.p.), did not modify the gastrointestinal transit in control mice (% transit: control 54±4; VDM11: 44±4, n=10, P>0.2) but further delayed gastrointestinal transit in acetic-acid treated mice (% transit: ileus 26±2, VDM 10±2, n=10, P<0.01). This effect was reversed (P<0.01) by a per se ineffective dose of SR141716A (1 mg/kg, i.p.).
CONCLUSIONS AND SIGNIFICANCE
We found that i.p. acetic acid administration to mice, which induces peritonitis, strongly inhibited intestinal motility, resulting in paralytic ileus. This pathological state was accompanied by significantly increased intestinal levels of anandamide compared with the small intestine of saline-treated mice. Anandamide is known to inhibit intestinal motility through activation of enteric CB1 receptors, which in most cases are expressed by cholinergic neurons. In the present study we have observed an increased number and density of CB1 receptors in myenteric plexus and nerve bundles and fibers of the external muscle of the jejunum in acid acetic-treated mice vs. controls. Such overexpression of CB1 receptors pairs with the increased levels of anandamide observed in acetic acid-treated mice, potentially resulting in overstimulation of the endocannabinoid system in myenteric neurons, which coexpress CB1 receptors with acetylcholine and substance P. CB1 receptor stimulation by various agonists, including anandamide, reduces acetylcholine release from enteric nerves and inhibits nonadrenergic noncholinergic excitatory transmission (which is mediated by the release of endogenous tachykinins such as substance P). Therefore, we propose that after peritonitis-induced ileus, overstimulation of CB1 receptors on acetylcholine/substance P-containing neurons leads to a reduced release of both neurotransmitters, with subsequent delayed motility.
To substantiate a causative role of the endogenous cannabinoid system in ileus, we carried out a series of pharmacological experiments. We first studied the effect of the selective CB1 receptor antagonist SR141716A. If the enhanced endocannabinoid tone observed in the small intestine of acetic acid-treated mice were causative of decreased intestinal motility, we should have observed an alleviation of this typical sign of paralytic ileus. This was indeed the case since SR141716A restored normal intestinal motility. This effect was likely mediated uniquely by cannabinoid CB1 (and not CB2) receptor blockade because it was counteracted by a per se ineffective dose of the selective cannabinoid CB1 receptor agonist ACEA, but not by the selective CB2 receptor agonist JWH-015. Moreover, the selective CB2 receptor antagonist SR144528 did not modify the acetic acid-induced decrease of intestinal motility. We found that SR141716A enhances small intestine transit in vehicle-treated mice. However, the antagonist was significantly more potent in control than in acetic acid-treated mice. This difference may be due to the increased levels of anandamide we observed in acetic acid-treated mice, since a greater amount of antagonist is thought to be needed to counteract the effect of higher amounts of anandamide on enteric CB1 receptors. Therefore, the lower potency of SR141716A in acetic acid-treated mice lends further, albeit indirect, support to our hypothesis that an enhanced anandamide tone is partly responsible for reduced intestinal motility during paralytic ileus.
To challenge this hypothesis further, we assessed whether pharmacological manipulation of anandamide levels with an inhibitor of its inactivation would result in an effect on intestinal motility during ileus. We reasoned that any agent that prolongs the effects of endogenous anandamide by selectively retarding its degradation would worsen the intestinal hypomotility in acetic acid-treated mice. We tested the selective inhibitor, the anandamide membrane transporter VDM11, on intestinal motility. We found that this compound delayed gastrointestinal transit in acetic acid-treated but not in control, mice, thus indicating not only a functional role of anandamide transport in terminating the biological action of anandamide during paralytic ileus but also the participation of endogenous anandamide in the reduction of motility during this pathological state.
In conclusion, we have shown that ileus-induced motility changes in response to peritoneal irritation is due at least in part to increased levels in the small intestine of anandamide, which contributes to reducing motility by acting on overexpressed enteric CB1 receptors. The causative role of anandamide in paralytic ileus is suggested not only by its increased levels during peritonitis, but by functional studies of motility with the blocker of anandamide uptake VDM11 (which worsens transit) and with the selective CB1 receptor antagonist SR141716A (which restores normal motility) (Fig. 2
). Our data not only provide new insights into the possible etiology of paralytic ileus, but open the possibility for the use of selective, nonpsychotropic CB1 receptor antagonists as new pharmacological tools for the clinical management of paralytic ileus.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0338fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0338fje 
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