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Department of Experimental Pharmacology, University of Naples ‘Federico II’, via D. Montesano 49, 80131 Naples Italy. E-mail: firstname.lastname@example.org:
Institute for the Chemistry of Molecules of Biological Interest, National Research Council, via Campi Flegrei 34, Ex-comprensorio Olivetti, Fabbricato 70, 80078, Pozzuoli, Napoli, Italy. E-mail: email@example.com
We have studied the effect of palmitoylethanolamide (PEA, 2.5 – 30 mg kg−1, i.p.) on upper gastrointestinal transit in control mice and in mice with chronic intestinal inflammation induced by croton oil.
PEA significantly and dose-dependently decreased intestinal transit. The inhibitory effect of PEA (10 mg kg−1) was not modified by the cannabinoid CB1 receptor antagonist SR141716A (0.3 mg kg−1, i.p.), the cannabinoid CB2 receptor antagonist SR144528 (1 mg kg−1, i.p.), NG-nitro-L-arginine methyl ester (L-NAME, 25 mg kg−1, i.p.), yohimbine (1 mg kg−1, i.p.), naloxone (2 mg kg−1, i.p.) or hexamethonium (1 mg kg−1, i.p.).
PEA levels were significantly decreased in the small intestine of croton oil-treated mice. In these animals, PEA also inhibited motility and this effect was not counteracted by SR141716A (0.3 mg kg−1), or SR144528 (1 mg kg−1).
Pre-treatment of mice with the amidase inhibitor phenylmethyl sulphonil fluoride (PMSF, 30 mg kg−1, i.p.) did not modify the inhibitory effect of PEA, either in control or in mice with inflammation.
It is concluded that PEA inhibits intestinal motility with a peripheral mechanism independent from cannabinoid receptor activation. The decreased levels of PEA in croton oil-treated might contribute, at least in part, to the exaggerated transit observed during chronic intestinal inflammation.
Male ICR mice (Harlan Italy, Corezzana, MI) (24 – 26 g) were used after 1 week of acclimation (temperature 23±2°C; humidity 60%). Food was withheld 6 h before transit measurement and 18 h before the induction of chronic intestinal inflammation. All animal experiments complied with the Italian D.L. n 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609/ECC).
Chronic intestinal inflammation
Inflammation was induced as previously described (Puig & Pol, 1998). Mice received orally two doses of croton oil (20 μl mouse−1) in 2 consecutive days. Motility was measured 4 days after the first administration of croton oil. This time was selected on the basis of a previous work (Puig & Pol, 1998), which reported that maximal inflammatory response occurred 4 days after the first treatment.
Upper gastrointestinal transit
Gastrointestinal transit was measured in control and in croton oil-treated mice. At this time, 0.1 ml 10 g mouse−1 of a black marker (10% charcoal suspension in 5% gum arabic) was administered orally to assess upper gastrointestinal transit as previously described (Puig & Pol, 1998). After 20 min the mice were killed by asphyxiation with CO2 and the gastrointestinal tract removed. The distance travelled by the marker was measured and expressed as a percentage of the total length of the small intestine from pylorus to caecum.
PEA (0.3 – 30 mg kg−1) or vehicle (ethanol, 15 μl mouse−1) were given intraperitoneally (i.p.) 20 min before charcoal administration, either to control mice or mice with chronic inflammation. In some experiments SR141716A (0.3 mg kg−1), SR144528 (1 mg kg−1), NG-nitro-L-arginine methyl ester (25 mg kg−1), yohimbine (1 mg kg−1), naloxone (2 mg kg−1), hexamethonium (1 mg kg−1) or phenilmethylsulphonyl fluoride (PMSF, 30 mg kg−1) were given (i.p.) before PEA administration (10 min before PEA for PMSF and 30 min before PEA for the other drugs). These doses were selected on the basis of previous published work (Santos & Rao, 1999; Izzo et al., 2001a; Wiley et al., 2000).
Identification and quantification of PEA
The small intestines of control and croton oil-treated mice were removed and tissue specimens were immediately weighed, immersed into liquid nitrogen, then stored at −70° until analysis. Tissue was then extracted with chloroform/methanol (2 : 1, by volume) containing 5 nmol d4-PEA, synthesized as described from d4-palmitic acid (Cambridge, U.K.) and ethanolamine (Bisogno et al., 1997). The lipid extracts were purified by silica column chromatography and normal phase high pressure liquid chromatography (NP-HPLC), carried out as described previously (Bisogno et al., 1997). The fraction corresponding to PEA (retention time 26 – 27 min) was derivatized and analysed as the tris-methyl-sylyl-derivatives by isotope dilution gas chromatography-mass spectrometry (GC-MS) carried out in the selected monitoring mode as described in detail elsewhere (Bisogno et al., 1999). Selected ions were at m/z=375 and 360, for d4-PEA, and at m/z=371 and 356, for endogenous PEA, and corresponded to the molecular ions or the loss of 15 mass units (loss of a methyl group). The areas of the peak at m/z=360 and 356 were used for quantitative measurements. Results were expressed as pmol mg−1 tissue.
Drugs used were: Palmitoylethanolamide (PEA) (Tocris Cookson, Bristol, U.K.), hexamethonium bromide, naloxone hydrochloride, NG-nitro-methyl arginine methyl ester (L-NAME) hydrochloride, phenylmethylsulphonyl fluoride (PMSF), yohimbine hydrochloride (SIGMA, Milan, Italy). SR141716A [(N-piperidin-1-yl)-5-(4-chlorophenyl)-1-2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride and SR144528 (N-[-1S-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide-3-carboxamide) were a gift from Dr Madaleine Mossé and Dr Francis Barth (SANOFI-Recherche, Montpellier, France). PEA was dissolved in ethanol, SR141716A and SR144528 in dimethyl sulphoxide (DMSO), while the other drugs were dissolved in saline.
Data are means±s.e.mean. To determine statistical significance, Student's t-test for unpaired data or one-way analysis of variance followed by Tukey-Kramer multiple comparisons test was used. A P-value less than 0.05 was considered significant. ED50 (the dose of PEA that produced 50% of maximal inhibition of motility) values were calculated using the computer program of Tallarida & Murray (1986).
Effect of PEA on motility in control mice
PEA (2.5 – 30 mg kg−1) inhibited the gastrointestinal propulsion of charcoal in control mice (Figure 1) (% inhibition of motility: 2.5 mg kg−1 4±3%, 5 mg kg−1 15±3%, 10 mg kg−1 46±3%, 20 mg kg−1 59±3%, 30 mg kg−1 59±2%). The inhibitory effect of PEA was significant starting from the dose of 10 mg kg−1. The inhibitory effect of PEA 10 mg kg−1 was not significantly modified by the CB1 receptor antagonist SR141716A (0.3 mg kg−1) or by the CB2 receptor antagonist SR144528 (1 mg kg−1) (Table 1). L-NAME (25 mg kg−1), yohimbine (1 mg kg−1), naloxone (2 mg kg−1) or hexamethonium (1 mg kg−1) also did not modify the inhibitory effect of PEA 10 mg kg−1 (Table 1). PMSF (30 mg kg−1) did not modify significantly the inhibitory effect of PEA (2.5 – 30 mg kg−1) on intestinal motility (ED50 values (mg kg−1) PEA 7.25±0.85; PMSF+PEA 7.55±0.91, n=9 – 10, P>0.2) (Figure 1).
Table 1. Effect of PEA (10 mg kg−1 i.p.) alone or in combination with various drugs on upper gastrointestinal transit in mice, either in control or in croton oil treated mice
In absence of any drugs (i.e. without PEA), SR141716A (0.3 mg kg−1), SR144528 (1 mg kg−1), L-NAME (25 mg kg−1), yohimbine (1 mg kg−1), naloxone (2 mg kg−1), hexamethonium (1 mg kg−1), PMSF (30 mg kg−1), at the dosage used, did not modify significantly gastrointestinal transit (% transit: control: 48±4, SR141716A 59±4, SR144528 49±3, L-NAME 44±3, yohimbine 50±4, naloxone 51±3, hexamethonium 51±3, PMSF 50±5, n=7 – 8 for each experimental group). Ethanol (15 μl mouse−1) or DMSO (10 μl mouse−1) (alone or in combination) did not modify significantly intestinal motility, either in control or in croton-oil treated mice (data not shown).
Effect of PEA on motility in croton oil-treated mice
As shown in Table 1, a charcoal meal administered intragastrically to control animals covered 48±3% of the total length of the small intestine in 20 min. Administration of croton oil (20 μl mouse−1 on 2 consecutive days, 3 and 4 days before charcoal administration) induced a significant increase in gastrointestinal transit (Table 1). Pre-treatment of the animals with PEA (3 – 30 mg kg−1) reversed the increase in gastrointestinal transit induced by croton oil in a dose-dependent fashion (Figure 2). The ED50 value for PEA calculated in mice with inflammation (7.44±0.89 mg kg−1) did not differ significantly (P>0.2, n=10) from the ED50 value calculated in control mice (7.25±0.85 mg kg−1). The inhibitory effect of PEA (10 mg kg−1) was not significantly modified either by SR141716A (0.3 mg kg−1) or by SR144528 (1 mg kg−1) (Table 1). As in the case of control mice, PMSF (30 mg kg−1) did not modify significantly the inhibitory effect of PEA (2.5 – 30 mg kg−1) on intestinal motility in mice with intestinal inflammation (Figure 2) (ED50 values (mg kg−1): croton oil: 7.44±0.89, croton oil+PMSF 7.03±0.77, n=10, P>0.2).
SR141716A (0.3 mg kg−1), SR144528 (1 mg kg−1) or PMSF (30 mg kg−1), administered alone, did not modify significantly gastrointestinal transit in croton oil-treated mice (data not shown).
PEA levels in the small intestine
Figure 3 shows that PEA was detected in the small intestine of control mice (77.5±6.3 pmol g−1 tissue) and that this production was significantly (P<0.05) decreased in the small intestine of croton oil-treated mice.
PEA is an endogenous fatty acid ethanolamide that shares some pharmacological actions with Δ9-tetrahydrocannabinol, the main ingredient of marijuana, and with the endocannabinoids anandamide and 2-arachidonylglycerol (Lambert & Di Marzo, 1999). However, PEA does not bind to cannabinoid CB1 and CB2 receptors and therefore it cannot be defined as an ‘endocannabinoid’ according to the definition given by di marzo (1998). Previous studies have shown that PEA possesses analgesic (Calignano et al., 1998), anti-inflammatory (Berdyshev et al., 1998) and central depressant activity (Adams et al., 1995). In the present study, we have revealed its presence in the small intestine and its ability to reduce intestinal motility, both during physiological conditions and in a pathological state.
Previous studies have shown that anandamide as well as other natural and synthetic cannabinoid agonists, by activating enteric CB1 receptors, can reduce intestinal motility either in vitro (Pertwee et al., 1995; Izzo et al., 1998) or in vivo (Fride, 1995; Calignano et al., 1997; Izzo et al., 2001a). In the present study we have shown that the selective cannabinoid CB1 receptor antagonist SR141716A, at doses able to counteract the inhibitory effect of anandamide (Izzo et al., 2001a), was not able to counteract the inhibitory effect of PEA on intestinal motility. There is also some evidence in literature that some effect of PEA can be mediated by as-yet uncharacterized ‘CB2-like’ receptors, because some pharmacological effects of PEA can be counteracted by the selective CB2 receptor antagonist SR144528 (Facci et al., 1995; Calignano et al., 1998). In the present study, however, the effect of PEA on intestinal motility was not modified by SR144528. The dose of SR144528 used in the present study was 10 fold higher than the dose of SR144528 able to counteract the analgesic effect of PEA (Calignano et al., 1998). Collectively, these results indicate that the effect of PEA on intestinal motility is not mediated by activation of cannabinoid receptors. Presynaptic/prejunctional systems, such as α2-adrenoceptors or opioid receptors, which, if activated, are known to inhibit intestinal motility, are not involved in the inhibitory effect of PEA. In fact, naloxone or yohimbine, antagonists of opioid or α2-adrenoceptors, respectively, failed to modify PEA-induced changes in motility. In addition, the effect of PEA was not modified by the ganglion blocker hexamethonium, thus suggesting a peripheral site of action. Moreover, it is unlikely that the inhibitory effect of PEA could derive from modulation of NO production, as pre-treatment of mice with the NO synthase inhibitor L-NAME did not modify PEA-induced changes in motility. Others have shown that PEA inhibits NO production in murine macrophages and that this effect does not appear to be mediated by cannabinoid receptors (Ross et al., 2000).
PMSF is a non-specific irreversible amidase inhibitor that inhibits the action of fatty acid amide hydrolase. Previous investigators have shown that PMSF enhanced the pharmacological activity of anandamide (Wiley et al., 2000; Lambert & Di Marzo, 1999), including its ability to reduce intestinal motility (Pertwee et al., 1995). In the present study, PMSF, at doses previously shown to be effective (Wiley et al., 2000), did not modify the inhibitory effect of PEA on intestinal motility. The lack of effect of PMSF is not surprising in the light of the observation the PEA is not hydrolyzed by fatty acid amide hydrolase as efficiently as anandamide (Lambert & Di Marzo, 1999), and that another amidase insensitive to PMSF has been identified for PEA (Ueda et al., 1999).
Mice with intestinal inflammation
Croton oil is an irritant that produces experimental chronic inflammation in the mouse small intestine. Inflammation is characterized by a clear disruption of the mucosa and an infiltration of lymphocyte in the submucosa (Puig & Pol, 1998). Macroscopic observation and increased wet weight, which is considered a reliable and sensitive indicator of the severity and extent of the inflammatory response, confirmed that inflammation occurred in our experimental conditions. Previous investigators have shown that the chronic intestinal inflammation induced by two consecutive doses of croton oil given 24 h apart (as in this study), produces maximal inflammatory response and maximal increase in gastrointestinal motility 4 days after the first dose of croton oil (Puig & Pol, 1998). Therefore the influence of PEA on intestinal motility, as well as the levels of PEA in the small intestine, were studied at this time point.
We have recently shown that chronic inflammation enhances the potency of cannabinoid receptor agonists on intestinal motility by up-regulating CB1 receptor expression in the small intestine (Izzo et al., 2001b). In the present study we have observed that PEA decreased intestinal motility in mice with inflammation, although with the same potency observed in control mice. In addition, consistent with the results obtained in control mice, the inhibitory effect of PEA was not mediated by activation of cannabinoid receptors and remained unchanged after pretreatment with the amidase inhibitor PMSF.
PEA has been detected in the rat brain, liver, skin, testis and skeletal muscle, in the canine heart and in mouse peritoneal macrophages, and it can be released following cellular injury (for review see Lambert & Di Marzo, 1999). Previous studies have also shown that PEA can be released with the endocannabinoid anandamide from different mammalian tissues (di marzo et al., 1994). We have recently detected anandamide in the small intestine of both control mice and in mice treated with croton oil (as in the present study) (Izzo et al., 2001b). However, no significant differences were observed in anandamide levels between control and inflamed mice (Izzo et al., 2001b). In the present study we have detected PEA in the small intestine of both control and inflamed mice and, in addition, we have observed a significant reduction of PEA level in the small intestine of croton oil-treated mice. The amounts of PEA that we found in the small intestine from control animals was higher than the levels previously described for anandamide in the same tissue (Izzo et al., 2001b) and in the high pmol g−1 tissue range of concentrations. If we assume an equal distribution of PEA throughout the small intestine, these amounts would correspond to an average concentration of about 70 nM, which would be not enough to influence the activity of CB1 or CB2 receptors, in agreement with the non-CB1, non-CB2 mechanism of action suggested here for exogenous PEA effect. In fact, some effect of PEA on cannabinoid receptors was only observed at concentrations >10 μM (Griffin et al., 2000). Nevertheless, this concentration of PEA might be sufficient to activate yet to be discovered sites of action for this compound, possibly involved in the control of intestinal motility. If this was the case, the decrease of PEA observed here in inflamed small intestine might contribute in part to the increased motility observed in croton oil-treated mice.
In the present study we have shown that PEA is able to inhibit intestinal motility in mice with a mechanism independent from cannabinoid receptor activation, under both physiological and pathological conditions. The decreased levels of PEA in croton oil-treated mice might contribute, at least in part, to the exaggerated transit observed during chronic inflammation. The previously undetected effect of PEA on intestinal motility broadens its pharmacological spectrum of actions and opens the possibility that this compound, which, unlike anandamide, has weak psychotropic effects, is used as a possible therapeutic drug for the treatment of intestinal hypermotility during inflammatory bowel diseases.
This work was supported by Cofinanziamento MURST and Enrico and Enrica Sovena Foundation (Rome, Italy) and MURST pr. 3933 (to V. Di Marzo). SR141716A and SR144528 were a kind gift from SANOFI (Montpellier, France).