In the present study we have evaluated the effect of CBC on intestinal motility in mice. CBC was evaluated on upper gastrointestinal transit (both in physiological and inflammatory conditions), colonic propulsion and whole gut transit in vivo . In vitro , we evaluated the effect of CBC on electrically or ACh‐induced contractions in the ileum. A preliminary account of this work has been communicated to the 20th Annual Symposium of the International Cannabinoid Research Society ( Romano et al ., 2010 ).

Methods

Animals Male ICR mice (Harlan Laboratories, S. Pietro al Natisone, Italy) weighing 20–25 g were used after a 1 week acclimatization period (temperature 23 ± 2°C; humidity 60%, free access to water and standard food). All animal care and experimental procedures complied with the principles of laboratory animal care (NIH publication no.86‐23, revised 1985) and the Italian D.L. no.116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609/ECC).

Intestinal inflammation Intestinal inflammation was induced as previously described (Pol and Puig, 1997; Capasso et al., 2008a). Briefly, two doses of croton oil (20 µL per mouse) for two consecutive days were orally administered to mice and four days after the first administration of croton oil, upper gastrointestinal transit of mice was measured. This time was selected on the basis of previous work (Pol and Puig, 1997), which reported that the maximal inflammatory response occurred 4 days after the first treatment.

Endocannabinoid extraction and measurement The duodenum, jejunum and ileum from control and croton oil‐treated mice (treated or not with CBC 15 mg·kg−1, i.p., 30 min before croton oil) were removed (4 days after the first administration of croton oil), and tissue specimens were immediately weighed, immersed into liquid nitrogen, and stored at −80°C until extraction of endocannabinoids. Tissues were extracted, purified and analysed as described in detail elsewhere (Di Marzo et al., 2008).

Quantitative (real‐time) RT‐PCR analysis The duodenum, jejunum and ileum from control and croton oil‐treated mice (treated or not with CBC 15 mg·kg−1, i.p., 30 min before croton oil) were removed (4 days after the first administration of croton oil) and collected in RNA later (Invitrogen, Carlsbad, CA, USA) and homogenized by a rotor‐stator homogenizer in 1.5 mL of Trizol® (Invitrogen). Total RNA was extracted according to the manufacturer's recommendations, dissolved in RNAase‐free water, and further purified by spin cartridge by the Micro‐to‐Midi total RNA purification system (Invitrogen). Total RNA was dissolved in RNA storage solution (Ambion, Austin, TX, USA), UV‐quantified by a Bio‐Photometer® (Eppendorf, Santa Clara, CA, USA), and stored at −80°C until use. RNA aliquots (6 µg) were digested by RNAse‐free DNAse I (Ambion DNA‐free™ kit) in a 20 µL final volume reaction mixture to remove residual contaminating genomic DNA. After DNAse digestion, concentration and purity of RNA samples were evaluated by the RNA‐6000‐Nano® microchip assay using a 2100 Bioanalyzer® equipped with a 2100 Expert Software® (Agilent, Santa Clara, CA, USA) following the manufacturer's instructions. For all samples tested, the RNA integrity number was greater than 8 relative to a 0–10 scale. One microgram of total RNA, as evaluated by the 2100 Bioanalyzer, was reverse‐transcribed in cDNA by the SuperScript III SuperMix (Invitrogen). The reaction mixture was incubated in a termocycler iCycler‐iQ5® (Bio‐Rad, Hercules, CA, USA) for a 5 min at 60°C step, followed by a rapid chilling for 2 min at 4°C. The protocol was stopped at this step and the reverse transcriptase was added to the samples, except the negative controls (–RT). The incubation was resumed with two thermal steps: 10 min at 25°C followed by 40 min at 50°C. Finally, the reaction was terminated by heating at 95°C for 10 min. Quantitative real‐time PCR was performed by an iCycler‐iQ5® in a 20µL reaction mixture containing 1 × SsoFast EVAGreen supermix (Bio‐Rad), 10 ng of cDNA (calculated on the basis of the retro‐transcribed RNA) and 330 nM for each primer. The amplification profile consisted of an initial denaturation of 2 min at 94°C and 40 cycles of 30 s at 94°C, annealing for 30 s at TaOpt (optimum annealing temperature, see following discussion) and elongation for 45 s at 68°C. Fluorescence data were collected during the elongation step. A final extension of 7 min was carried out at 72°C, followed by melt‐curve data analysis. Assays were performed in quadruplicate (maximum ΔCt of replicate samples <0.5), and a standard curve from consecutive fivefold dilutions (100 to 0.16 ng) of a cDNA pool representative of all samples was included for PCR efficiency determination. Optimized primers for SYBR‐green analysis and optimum annealing temperatures were designed by the Allele‐Id software version 7.0 (Biosoft International, Palo Alto, CA, USA) and were synthesized (HPLC‐purification grade) by MWG‐Biotech (NAPE‐PLD accession NM_178728, F: CGCTGATGGTGGAAATGG, R: GTGGTTGTGACTGATGAGG; CB 1 accession NM_007726, F: CTACCTGATGTTCTGGAT, R: GTGTGAATGATGATGCTT; CB 2 accession, F: ATCTCCTCTCACTCACTTATCTG, R: GGTTTCTTGCTCTCACACTTT; TRPA1 accession NM_177781, F: GGAGATATGTGTAGATTAGAAGAC, R: TCGGAGGTTTGGATTTGC; GDE1 accession NM_019580.4, F: ATAACACAGTAGATAGGACAACA, R: AGCAGCAGAAGCCATATC; FAAH accession NM_010173, F: GCCTCAAGGAATGCTTCA, R: AGTCACTCTCCGATGTCA). Relative expression calculation – to correct for PCR efficiency and normalized with respect to reference gene β‐actin (accession: NM_007393; F: CCAGGCATTGCTGACAGG; R: TGGAAGGTGGACAGTGAGG) and HPRT (accession: NM_013556; F: TTGACACTGGTAAAACAATGC; R: GCCTGTATCCAACACTTCG) – was performed by iQ5 software. Results are expressed as fold expression, compared with control (=1) (Izzo et al., 2008).

Upper gastrointestinal transit in vivo Transit was measured by evaluating the intestinal location of rhodamine‐B‐labelled dextran (Izzo et al., 2009b). Animals were given fluorescent‐labelled dextran (100 µL of 25 mg·mL−1 stock solution) via a gastric tube into the stomach. At 20 min after administration, the animals were killed by asphyxiation with CO 2 and the entire small intestine with its contents was divided into 10 equal parts. The intestinal contents of each bowel segment were vigorously mixed with 2 mL of saline solution to obtain a supernatant containing the rhodamine. The supernatant was centrifuged at 35 x g to precipitate the intestinal chyme. The fluorescence in duplicate aliquots of the cleared supernatant was read in a multi‐well fluorescence plate reader (LS55 Luminescence spectrometer, Perkin‐Elmer Instruments, Waltham, MA, USA; excitation 530 ± 5 nm and emission 590 ± 10 nm) for quantification of the fluorescent signal in each intestinal segment. From the distribution of the fluorescent marker along the intestine, we calculated the geometric centre (GC) of small intestinal transit as follows: GC¼S (fraction of fluorescence per segment·segment number−1) GC ranged from 1 (minimal motility) to 10 (maximal motility). CBC (1–20 mg·kg−1), or vehicle was given (i.p.) 30 min before the oral administration of the fluorescent marker, both to control mice and to mice with intestinal inflammation induced by croton oil. In croton oil‐treated animals, the effect of CBC (10 mg·kg−1) was evaluated in animals pretreated (i.p., 10 min before CBC) with the CB 1 receptor antagonist rimonabant (0.1 mg·kg−1), the CB 2 receptor antagonist SR144528 (1 mg·kg−1) or the selective TRPA1 antagonists HC‐030031 (30 mg·kg−1) and AP18 (100 mg·kg−1). The doses of the cannabinoid receptor antagonists used have been previously shown in our laboratory to counteract the effect of selective cannabinoid receptor agonists on croton‐oil‐ induced hypermotility in mice (Capasso et al., 2008b). The dose of HC‐030031 (30 mg·kg−1) was selected based on previous work (McNamara et al., 2007), in which it was shown that this antagonist, given i.p., attenuated TRPA1‐mediated pain in mice. Higher doses of HC‐030031 were not used because they tend to increase, given alone, upper gastrointestinal transit (data not shown). On the other hand, AP18, even at the high dose of 100 mg·kg−1, given alone, did not affect transit.

Colonic propulsion in vivo Distal colonic propulsion was measured as previously described (Broccardo et al., 1998; Borrelli et al., 2006). A single 3 mm glass bead was inserted 2 cm into the distal colon of each mouse with the aid of a catheter and the time to expulsion of the glass bead was determined for each animal. CBC (10 and 20 mg·kg−1), WIN 55,212‐2 (1 mg·kg−1, used as a positive control) or vehicle was given (i.p.) 30 min before glass bead insertion.

Whole gut transit time in vivo Mice were housed in individual cages 72 h before the experiment. On the day of the experiment, they were acclimatized to an empty cage (devoid of bedding) for 1 h before drug treatment. Thirty minutes after i.p. administration of CBC (10 and 20 mg·kg−1), vehicle or the cannabinoid receptor agonist WIN 55,212‐2 (1 mg·kg−1, used as a positive control), mice received by gastric gavage 0.2 mL of 6% carmine red suspension in 0.5% carboxymethylcellulose. The time to the first red bowel movement was measured in min and constituted the whole gut transit time (Storr et al., 2010).

Electrically (and agonists)‐induced contractions in the isolated ileum Mice were killed by asphyxiation with carbon dioxide and the ileum was removed, flushed of luminal contents, and placed in Krebs solution (composition: NaCl 119 mM, KCl 4.75 mM, KH2PO4 1.2 mM, NaHCO3 25 mM, MgSO 4 1.5 mM, CaCl 2 2.5 mM, and glucose 11 mM). Segments of 1.0–1.5 cm were cut from the distal ileum and placed in 20 mL thermostatically controlled (37°C) organ bath containing Krebs solution gassed with 95% O 2 and 5% CO 2 . The tissues were connected to an isometric transducer (tension: 5 mN) in such a way as to record contractions from the longitudinal axis. Mechanical activity was digitized on an analogue‐to‐digital converter, visualized, recorded and analysed on a personal computer using the PowerLab/400 system (Ugo Basile, Comerio, Italy). All experiments started after a minimal 1 h equilibration period. Contractions to electrical field stimulation (EFS; 8 Hz for 10 s, 400 mA, 1 ms pulse duration) were obtained by a pair of electrodes placed around the ileal tissue derived from both control and croton oil‐treated animals; the interval between each contraction was 20 min. EFS‐induced contractions were performed in the presence of the acetylcholinesterase inhibitor neostigmine (1 µM), to potentiate cholinergic neurotransmission (Baldassano et al., 2009). After stable control contractions evoked by EFS had been recorded, the contractile responses were observed in the presence of increasing cumulative concentrations of CBC (10−8–10−4 M). The contact time for each concentration was 20 min. Preliminary experiments showed that this contact time was sufficient for CBC to achieve maximal pharmacological effect. The effect of CBC on EFS‐induced contractions was also evaluated after the administration in the bath (contact time ≥30 min) of the non‐selective channel‐blocker ruthenium red (3 × 10−6 M), the selective TRPA1 HC‐030031 (10−5 M), the cannabinoid CB 1 receptor antagonist rimonabant (3 × 10−8 M), the CB 2 receptor antagonist SR144528 (10−7 M), L‐NAME (3 × 10−4 M) plus apamin (10−7 M) (alone or in combination), ω‐conotoxin (10−8 M), the non‐selective PDE inhibitor IBMX (10−7 M), the cAMP‐selective PDE inhibitor rolipram (10−6 M) or the cell‐permeable activator of AC, forskolin (10−7 M). The concentration of rimonabant (3 × 10−8 M) was able to counteract the inhibitory effect of the cannabinoid receptor agonist WIN55,212‐2 on EFS‐induced contractions (data not shown). The concentrations of HC‐030031 (10−5 M) and ruthenium red (3 × 10−6 M) were approximately two‐three fold higher than the IC 50 value calculated for these compounds as TRPA1 antagonists (McNamara et al., 2007; Alexander et al., 2011). Higher concentrations of the two TRPA1 antagonists were not used because they inhibited, per se, the EFS‐induced‐induced contractions. The other concentrations used in the present study were selected on the basis of previous work (Coutts and Pertwee, 1998; Nocerino et al., 2002; Capasso et al., 2008b; Borrelli et al., 2011). In a separate set of experiments, the effect of the selective cannabinoid agonist WIN5555,212‐2 (10−9–10−6 M, contact time for each concentration: 20 min) on EFS‐induced contractions was also evaluated [alone or in the presence of ω‐conotoxin (10−8 M), IBMX (10−7 M), rolipram (10−6 M) or forskolin (10−7 M)]. In some experiments, the effect of CBC (10−8–10−4 M) was also evaluated (contact time 20 min) on the contractions produced by exogenous ACh (10−6 M) or KCl (10−2 M). ACh or KCl was left in contact with the tissue for 60 and 90 s, respectively, and then washed out. In one set of experiments, the effect of CBC on ACh‐induced contractions was evaluated in the presence (contact time ≥30 min) of cyclopiazonic acid (CPA; 10−5 M, a sarcoplasmic reticulum Ca2+ inhibitor), verapamil (10−6 M) (a L‐type Ca2+ blocker) or ω‐conotoxin (10−8 M). In this set of experiments, we also evaluated the effect of eugenol (10−7–3 × 10−4 M, contact time for each concentration: 20 min) on ACh‐induced contractions. Contractions are expressed as % of contractions produced by 10−3 M ACh; this concentration of ACh produced a maximal contractile response (100% contraction).

Statistics Data are expressed as the mean ± SEM of experiments in n mice. To determine statistical significance, Student's t‐test was used for comparing a single treatment mean with a control mean, and a one‐way ANOVA followed by a Tukey–Kramer multiple comparisons test was used for analysis of multiple treatment means. P‐values < 0.05 were considered significant.