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• W2093229075 abstract "Background & Aims: Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis both of the endocannabinoids (which are known to inhibit intestinal motility) and other bioactive amides (palmitoylethanolamide, oleamide, and oleoylethanolamide), which might affect intestinal motility. The physiologic role of FAAH in the gut is largely unexplored. In the present study, we evaluated the possible role of FAAH in regulating intestinal motility in mice in vivo. Methods: Motility was measured by evaluating the distribution of a fluorescent marker along the small intestine; FAAH messenger RNA (mRNA) levels were analyzed by reverse-transcription polymerase chain reaction (RT-PCR); endocannabinoid levels were measured by isotope-dilution, liquid chromatography, mass spectrometry. Results: Motility was inhibited by N-arachidonoylserotonin (AA-5-HT) and palmitoylisopropylamide, 2 selective FAAH inhibitors, as well as by the FAAH substrates palmitoylethanolamide, oleamide, and oleoylethanolamide. The effect of AA-5-HT was reduced by the CB1 receptor antagonist rimonabant and by CB1 deficiency in mice but not by the vanilloid receptor antagonist 5′-iodoresiniferatoxin. In FAAH-deficient mice, pharmacologic blockade of FAAH did not affect intestinal motility. FAAH mRNA was detected in different regions of the intestinal tract. Conclusions: We conclude that FAAH is a physiologic regulator of intestinal motility and a potential target for the development of drugs capable of reducing intestinal motility. Background & Aims: Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis both of the endocannabinoids (which are known to inhibit intestinal motility) and other bioactive amides (palmitoylethanolamide, oleamide, and oleoylethanolamide), which might affect intestinal motility. The physiologic role of FAAH in the gut is largely unexplored. In the present study, we evaluated the possible role of FAAH in regulating intestinal motility in mice in vivo. Methods: Motility was measured by evaluating the distribution of a fluorescent marker along the small intestine; FAAH messenger RNA (mRNA) levels were analyzed by reverse-transcription polymerase chain reaction (RT-PCR); endocannabinoid levels were measured by isotope-dilution, liquid chromatography, mass spectrometry. Results: Motility was inhibited by N-arachidonoylserotonin (AA-5-HT) and palmitoylisopropylamide, 2 selective FAAH inhibitors, as well as by the FAAH substrates palmitoylethanolamide, oleamide, and oleoylethanolamide. The effect of AA-5-HT was reduced by the CB1 receptor antagonist rimonabant and by CB1 deficiency in mice but not by the vanilloid receptor antagonist 5′-iodoresiniferatoxin. In FAAH-deficient mice, pharmacologic blockade of FAAH did not affect intestinal motility. FAAH mRNA was detected in different regions of the intestinal tract. Conclusions: We conclude that FAAH is a physiologic regulator of intestinal motility and a potential target for the development of drugs capable of reducing intestinal motility. The endogenous cannabinoid system includes cannabinoid (CB1 and CB2) receptors, their endogenous ligands (the endocannabinoids), and the enzymes for the synthesis and inactivation of these ligands.1Howlett A.C. Barth F. Bonner T.I. Cabral G. Casellas P. Devane W.A. Felder C.C. Herkenham M. Mackie K. Martin B.R. Mechoulam R. Pertwee R.G. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors.Pharmacol Rev. 2002; 54: 161-202Crossref PubMed Scopus (2270) Google Scholar, 2Di Marzo V. Bifulco M. De Petrocellis L. The endocannabinoid system and its therapeutic exploitation.Nat Rev Drug Disc. 2004; 3: 771-784Crossref PubMed Scopus (867) Google Scholar The endocannabinoids anandamide and 2-arachidonylglycerol (2-AG) may reduce gastrointestinal motility through activation of enteric CB1 receptors; potential therapeutic applications of this activity include the treatment of motility disorders such as gastroesophageal reflux disease, irritable bowel syndrome, diarrhea, and inflammatory bowel diseases.3Coutts A.A. Izzo A.A. The gastrointestinal pharmacology of cannabinoids An update.Curr Opin Pharmacol. 2004; 4: 572-579Crossref PubMed Scopus (88) Google Scholar, 4Hornby P.J. Prouty S.M. Involvement of cannabinoid receptors in gut motility and visceral perception.Br J Pharmacol. 2004; 141: 1335-1345Crossref PubMed Scopus (88) Google Scholar Several experiments have demonstrated that the CB1 receptor antagonist rimonabant (SR141716A), in the absence of any exogenous agonist, produces motility changes that are invariably opposite in direction to those caused by the cannabinoid receptor agonists. For example, rimonabant is known to increase (1) electrically induced contractions and peristalsis in isolated intestinal segments from rodents,5Pertwee R.G. Fernando S.R. Nash J.E. Coutts A.A. Further evidence for the presence of cannabinoid CB1 receptors in guinea-pig small intestine.Br J Pharmacol. 1996; 118: 2199-2205Crossref PubMed Scopus (160) Google Scholar, 6Izzo A.A. Mascolo N. Borrelli F. Capasso F. Excitatory transmission to the circular muscle of the guinea-pig ileum evidence for the involvement of cannabinoid CB1 receptor.Br J Pharmacol. 1998; 124: 1363-1368Crossref PubMed Scopus (100) Google Scholar, 7Begg M. Dale N. Llaudet E. Molleman A. Parsons M.E. Modulation of the release of endogenous adenosine by cannabinoids in the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum.Br J Pharmacol. 2002; 137: 1298-1304Crossref PubMed Scopus (39) Google Scholar, 8Mancinelli R. Fabrizi A. Del Monaco S. Azzena G.B. Vargiu R. Colombo G.C. Gessa G.L. Inhibition of peristaltic activity by cannabinoids in the isolated distal colon of mouse.Life Sci. 2001; 69: 101-111Crossref PubMed Scopus (42) Google Scholar (2) occurrence of transient lower esophageal sphincter relaxation in dogs,9Lehmann A. Blackshaw L.A. Branden L. Carlsson A. Jensen J. Nygren E. Smid S.D. Cannabinoid receptor agonism inhibits transient lower esophageal sphincter relaxations and reflux in dogs.Gastroenterology. 2002; 123: 1129-1134Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar and (3) intestinal motility in mice in vivo, both in the small10Izzo A.A. Mascolo N. Borrelli F. Capasso F. Defecation, intestinal fluid accumulation and motility in rodents implications of cannabinoid CB1 receptors.Naunyn Scmideberg’s Arch Pharmacol. 1999; 359: 65-70Crossref PubMed Scopus (77) Google Scholar, 11Casu M.A. Porcella A. Ruiu S. Saba P. Marchese G. Carai M.A.M. Reali R. Gessa G.L. Pani L. Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract.Eur J Pharmacol. 2003; 459: 97-105Crossref PubMed Scopus (80) Google Scholar and in the large12Pinto L. Izzo A.A. Cascio M.G. Bisogno T. Hospodar-Scott K. Brown D.R. Mascolo N. Di Marzo V. Capasso F. Endocannabinoids as physiological regulators of colonic propulsion in mice.Gastroenterology. 2002; 123: 227-234Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar intestine. These effects cannot be attributed unequivocally to the displacement of endogenous cannabinoids because rimonabant may behave as an inverse agonist at CB1 receptors in vitro.13MacLennan S.J. Reynen P.H. Kwan J. Bonhaus D.W. Evidence for inverse agonism of SR141716A at human recombinant cannabinoid CB1 and CB2 receptors.Br J Pharmacol. 1998; 124: 619-622Crossref PubMed Scopus (167) Google ScholarInactivation of endocannabinoid signaling is dependent on cellular uptake, localization to appropriate intracellular compartments, and enzymatic hydrolysis. The latter reaction produces arachidonic acid and either ethanolamine (from anandamide) or glycerol (from 2-AG).14Deutsch D.G. Ueda N. Yamamoto S. The fatty acid amide hydrolase (FAAH).Prostaglandins Leukot Essent Fatty Acids. 2002; 66: 201-210Abstract Full Text PDF PubMed Scopus (186) Google Scholar Although it is generally recognized that there is uptake, intracellular transport, and hydrolysis of anandamide, only the latter step has been conclusively assigned to a protein, the fatty acid amide hydrolase (FAAH).2Di Marzo V. Bifulco M. De Petrocellis L. The endocannabinoid system and its therapeutic exploitation.Nat Rev Drug Disc. 2004; 3: 771-784Crossref PubMed Scopus (867) Google Scholar, 14Deutsch D.G. Ueda N. Yamamoto S. The fatty acid amide hydrolase (FAAH).Prostaglandins Leukot Essent Fatty Acids. 2002; 66: 201-210Abstract Full Text PDF PubMed Scopus (186) Google Scholar FAAH is a membrane-associated protein that is localized to internal membranes, such as the endoplasmic reticulum, at which it is active. The broad substrate specificity of FAAH allows it to catalyze the hydrolysis not only of the endocannabinoids anandamide and 2-AG but also of palmitoylethanolamide (PEA), oleamide (a sleep-inducing factor),15Cravatt B.F. Prospero-Garcia O. Siuzdak G. Gilula N.B. Henriksen S.J. Boger D.L. Lerner R.A. Chemical characterization of a family of brain lipids that induce sleep.Science. 1995; 268: 1506-1509Crossref PubMed Scopus (532) Google Scholar and oleoylethanolamide, whose biologic effects may be independent of CB1 receptors.16Lambert D.M. Di Marzo V. The palmitoylethanolamide and oleamide enigmas are these two fatty acid amides cannabimimetic?.Curr Med Chem. 1999; 6: 757-773PubMed Google Scholar, 17Fu J. Gaetani S. Oveisi F. Lo Verme J. Serrano A. Rodriguez De Fonseca F. Rosengarth A. Luecke H. Di Giacomo B. Tarzia G. Piomelli D. Oleylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-α.Nature. 2003; 425: 90-93Crossref PubMed Scopus (887) Google Scholar FAAH activity has been detected in the rodent intestine and was found to be increased in the croton oil model of intestinal inflammation.18Izzo A.A. Fezza F. Capasso R. Bisogno T. Pinto L. Iuvone T. Esposito G. Mascolo N. Di Marzo V. Capasso F. Cannabinoid CB1-receptor mediated regulation of gastrointestinal motility in mice in a model of intestinal inflammation.Br J Pharmacol. 2001; 134: 563-570Crossref PubMed Scopus (226) Google Scholar However, to date, selective FAAH inhibitors have not been evaluated in the gastrointestinal tract.The present study investigates the possible role of FAAH in the control of intestinal motility in mice in vivo. To this end, we used the selective FAAH inhibitors N-arachidonoylserotonin (AA-5-HT)19Bisogno T. Melck D. De Petrocellis L. Bobrov M.Y.U. Gretskaya N.M. Bezuglov V.V. Sitachitta N. Gerwick W.H. Di Marzo V. Arachidonoylserotonin and other novel inhibitors of fatty acid amide hydrolase.Biochem Biophys Res Commun. 1998; 248: 515-522Crossref PubMed Scopus (184) Google Scholar and palmitoylisopropylamide (PIP)20Jonsson K.O. Vandevoorde S. Lambert D.M. Tiger G. Fowler C.J. Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide.Br J Pharmacol. 2001; 133: 1263-1275Crossref PubMed Scopus (136) Google Scholar as well as FAAH-deficient mice. In addition, we report the distribution of FAAH messenger RNA (mRNA) along the mouse intestinal tract.Materials and MethodsAnimalsMale ICR mice (Harlan Italy, Corezzana, MI) (20–22 g) were normally used, but, in our preliminary experiments, some female ICR mice were studied as well. No difference in sensitivity to FAAH inhibitors was found between males and females. Mice lacking CB1 receptor and FAAH genes were generated and genotyped as previously described.21Marsicano G. Wotjak C.T. Azad S.C. Bisogno T. Rammes G. Cascio M.G. Hermann H. Tang J. Hofmann C. Zieglgansberger W. Di Marzo V. Lutz B. The endogenous cannabinoid system controls extinction of aversive memories.Nature. 2002; 418: 530-534Crossref PubMed Scopus (1418) Google Scholar, 22Cravatt B.F. Demarest K. Patricelli M.P. Bracey M.H. Giang D.K. Martin B.R. Lichtman A.H. Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase.Proc Natl Acad Sci U S A. 2001; 98: 9371-9376Crossref PubMed Scopus (1094) Google Scholar Female homozygous wild-type and homozygous mutant littermates (19–22 g) were used in the experiments. Mutant mice were in a mixed genetic background with a predominance of C57BL/6N contribution (5 backcrosses for both mutant lines). Mice were fed ad libitum with standard mouse food, except for the 12-hour period immediately preceding the experiments.Functional StudiesTransit was measured by evaluating the intestinal location of rhodamine-B–labeled dextran.23Kalff J.C. Buchholz B.M. Eskandari M.K. Hierholzer C. Schraut W.H. Simmons R.L. Bauer A.J. Biphasic response to gut manipulation and temporal correlation of cellular infiltrates and muscle dysfunction in rat.Surgery. 1999; 126: 498-509Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 24Schwarz N.T. Kalff J.C. Turler A. Engel B.M. Watkins S.C. Billiar T.R. Bauer A.J. Prostanoid production via COX-2 as a causative mechanism of rodent postoperative ileus.Gastroenterology. 2001; 121: 1354-1371Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar Animals were given fluorescent-labeled dextran (100 μL of 25 mg/mL stock solution) via a gastric tube into the stomach. Twenty minutes after administration, the entire small intestine with its content was divided into 10 equal parts. The intestinal contents of each bowel segment were vigorously mixed with 2 mL saline solution to obtain a supernatant containing the rhodamine. The supernatant was centrifuged at 500 rpm to force the intestinal chime to a pellet. The fluorescence in duplicate aliquots of the cleared supernatant was read in a multiwell fluorescence plate reader (LS55 Luminescence spectrometer; Perkin Elmer Instruments; 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 center (GC) of small intestinal transit as follows:GC = ∑(fraction ⁢of fluorescence⁢per⁢segment×segmentnumber) GC ranged from 1 (minimal motility) to 10 (maximal motility).25Shook J.E. Burks T.F. Psychoactive cannabinoids reduce gastrointestinal propulsion and motility in rodents.J Pharmacol Exp Ther. 1989; 249: 444-449PubMed Google Scholar This procedure yielded an accurate, nonradioactive measurement of intestinal transit.24Schwarz N.T. Kalff J.C. Turler A. Engel B.M. Watkins S.C. Billiar T.R. Bauer A.J. Prostanoid production via COX-2 as a causative mechanism of rodent postoperative ileus.Gastroenterology. 2001; 121: 1354-1371Abstract Full Text Full Text PDF PubMed Scopus (217) Google ScholarDrug AdministrationN-arachidonoylserotonin (AA-5-HT, 1–20 mg/kg), palmitoylisopropylamide (PIP, 1–20 mg/kg), oleamide (1–20 mg/kg), oleoylethanolamide (1–20 mg/kg), palmitoylethanolamide (PEA; 1–20 mg/kg), or vehicle were given intraperitoneally (IP) 30 minutes before the administration of the fluorescent marker. In some experiments, rimonabant (0.1 mg/kg), 5′-iodoresiniferatoxin (I-RTX; 0.75 mg/kg), or SR144528 (1 mg/kg) were given IP 10 minutes before AA-5-HT (15 mg/kg). Rimonabant (0.1 mg/kg) was also given 10 minutes before the administration of PEA, oleamide, or oleoylethanolamide (all at the dose of 10 mg/kg). I-RTX and SR144528 doses were selected on the basis of previous work.26Rigoni M. Trevisani M. Gazzieri D. Nadaletto R. Tognetto M. Creminon C. Davis J.B. Campi B. Amadesi S. Geppetti P. Harrison S. Neurogenic responses mediated by vanilloid receptor-1 (TRPV1) are blocked by the high affinity antagonist, iodo-resiniferatoxin.Br J Pharmacol. 2003; 138: 977-985Crossref PubMed Scopus (95) Google Scholar, 27Izzo A.A. Capasso F. Costagliola A. Bisogno T. Marsicano G. Ligresti A. Matias I. Capasso R. Pinto L. Borrelli F. Cecio A. Lutz B. Mascolo N. Di Marzo V. An endogenous cannabinoid tone attenuates cholera toxin-induced fluid accumulation in mice.Gastroenterology. 2003; 125: 765-774Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 28Buckley M.J. Surowy C. Meyer M. Curzon P. Mechanism of action of A-85380 in an animal model of depression.Prog Neuropsychopharmacol Biol Psychiatry. 2004; 28: 723-730Crossref PubMed Scopus (36) Google Scholar In some experiments, the effect of IP-injected anandamide (1–20 mg/kg), PEA (1–20 mg/kg), or loperamide (0.03–3 mg/kg) was evaluated 30 minutes after the administration of AA-5-HT (5 mg/kg, IP)Identification and Quantification of Endocannabinoids and PalmitoylethanolamideFull-thickness small intestines from mice given (IP) vehicle, AA-5-HT (1–15 mg/kg), oleamide (15 mg/kg), or oleoylethanolamide (15 mg/kg), as well as from FAAH and wild-type deficient mice, were removed, and tissue specimens were immediately weighed, immersed into liquid nitrogen, and stored at −70°C until chromatographic separation of endocannabinoids. Tissues were extracted with chloroform/methanol (2:1, by volume) containing each of 200 pmol d8-anandamide, d4-palmitoylethanolamide, and d5-2-AG, synthesized as described previously (for the former compounds),29Bisogno T. Berrendero F. Ambrosino G. Cebeira M. Ramos J.A. Fernandez-Ruiz J.J. Di Marzo V. Brain regional distribution of endocannabinoids implications for their biosynthesis and biological function.Biochem Biophys Res Commun. 1999; 256: 377-380Crossref PubMed Scopus (271) Google Scholar or provided by Cayman Chemicals (for d5-2-AG, Ann Arbor, MI). The lipid extracts were purified by silica column chromatography, carried out as described previously,29Bisogno T. Berrendero F. Ambrosino G. Cebeira M. Ramos J.A. Fernandez-Ruiz J.J. Di Marzo V. Brain regional distribution of endocannabinoids implications for their biosynthesis and biological function.Biochem Biophys Res Commun. 1999; 256: 377-380Crossref PubMed Scopus (271) Google Scholar and the fractions containing anandamide, palmitoylethanolamide, and 2-AG were analyzed by isotope-dilution, liquid chromatography, atmospheric pressure, chemical ionization mass spectrometry (LC-APCI-MS) carried out in the selected monitoring mode as described in detail elsewhere.21Marsicano G. Wotjak C.T. Azad S.C. Bisogno T. Rammes G. Cascio M.G. Hermann H. Tang J. Hofmann C. Zieglgansberger W. Di Marzo V. Lutz B. The endogenous cannabinoid system controls extinction of aversive memories.Nature. 2002; 418: 530-534Crossref PubMed Scopus (1418) Google Scholar Results were expressed as pmol or nmol per g of wet tissue. Because, during tissue extraction/purification, both d8- and native 2-AG are partly transformed into the 1(3)-isomers and only a limited amount of arachidonic acid is present on the sn-1(3) position of (phospho)glycerides, the amounts of 2-AG reported here represent the combined mono-arachidonyl-glycerol peaks.Semiquantitative RT-PCR for FAAH mRNATotal RNA from both the small (duodenum, jejunum, and ileum) and the large (proximal and distal colon) intestine of each animal was extracted using Trizol reagent according to the manufacturer’s recommendations (GibcoBRL). Following extraction, RNA was precipitated using ice-cold isopropanol, resuspended in diethyl pyrocarbonate-treated water (Sigma). The integrity of RNA was verified following separation by electrophoresis into a 1% agarose gel containing ethidium bromide. RNA was treated with RNAse-free DNAse I (Ambion DNA-free kit) according to the manufacturer’s recommendations, to digest contaminating genomic DNA. Subsequently DNAse and divalent cations were removed.The expression of mRNA for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and FAAH was examined by reverse transcription (RT) coupled to the polymerase chain reaction (PCR). Total RNA was reverse transcribed using oligo dT primers. DNA amplifications were carried out in PCR buffer (Q-Biogen) containing 2 μL cDNA, 500 μmol/L dNTP, 2 mmol/L MgCl2, 0.8 μmol/L each primer, and 0.5 U Taq polymerase (Q-Biogen). The thermal reaction profile consisted of a denaturation step at 94°C for 1 minute, annealing at 60°C for 1 minute, and an extension step at 72°C for 1 minute. A final extension step of 10 minutes was carried out at 72°C. Thirty PCR cycles were observed to be optimal and in the linear portion of the amplification curve (data not shown). The reaction was performed in a PE Gene Amp PCR System 9600 (Perkin Elmer). After reaction, the PCR products were electrophoresed on a 2% agarose gel containing ethidium bromide for UV visualization.The specific oligonucleotides were synthesized on the basis of cloned cDNA sequences of GAPDH, FAAH, and CB1 common to the rat and mouse. For GAPDH, the primers sequences were 5′-CCCTTCATTGACCTCAACTACATGGT-3′ (nt 208–233; sense) and 5′-GAGGGCCATCCACAGTCTTCTG-3′ (nt 655–677; antisense, accession No. AH007340 ). The FAAH sense and antisense primers were 5′-GTGGTGCT(G/A)ACCCCCATGCTGG-3′ (nt 1407–1428) and 5′-TCCACCTCCCGCATGAACCGCAGACA-3′ (nt 1683–1708, accession No. AF098010 ). The CB1 sense and antisense primers were 5′-GATGTCTTTGGGAAGATGAACAA GC-3′ (nt 1095–1119) and 5′-AGACGTGTCTGTGGACACAGACATGG-3′ (nt 1380–1405). The expected sizes of the amplicons were 470 bp for GAPDH, 300 bp for FAAH, and 309 bp for CB1. The expression of the housekeeping gene GAPDH was used as an internal standard. No PCR products were detected when the reverse transcriptase step was omitted (data not shown).DrugsN-Arachidonoylserotonin (AA-5-HT) was synthesized as described previously.19Bisogno T. Melck D. De Petrocellis L. Bobrov M.Y.U. Gretskaya N.M. Bezuglov V.V. Sitachitta N. Gerwick W.H. Di Marzo V. Arachidonoylserotonin and other novel inhibitors of fatty acid amide hydrolase.Biochem Biophys Res Commun. 1998; 248: 515-522Crossref PubMed Scopus (184) Google Scholar PIP, oleamide, oleoylethanolamide, PEA, anandamide, and I-RTX were purchased from Tocris Cookson (Bristol, United Kingdom), loperamide hydrochloride from Sigma (Milan, Italy). Rimonabant (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) were a kind gift from Drs. Madaleine Mossè and Francis Barth (SANOFI-Recherche, Montpellier, France).AA-5-HT and palmitoylisopropylamide were dissolved in DMSO/Tween 80 (1:4), oleamide and palmitoylethanolamide in ethanol (4 μL/mouse), oleoylethanolamide and iodoresiniferatoxin in DMSO, anandamide in Tocrisolve (soya oil/water [1:4 emulsion]), and loperamide in 2% DMSO. The drug vehicles (20 μL/mouse of DMSO/Tween 80, 4 μL/mouse DMSO, 4 μL/mouse ethanol, 40 μL/mouse Tocrisolve, or 50 μL/mouse 2% DMSO) had no effect on intestinal motility.ResultsMotilityIntraperitoneal administration of AA-5-HT (1–20 mg/kg; Figure 1A) and PIP (1–20 mg/kg; Figure 1B) produced a dose-dependent inhibition of transit. Both compounds gave rise to significant inhibitory effects for the 10-mg/kg dose. A per se noneffective dose of the CB1 receptor antagonist rimonabant (0.1 mg/kg), but not the CB2 receptor antagonist SR144528 (1 mg/kg, IP) nor the vanilloid receptor antagonist I-RTX (0.75 mg/kg, IP), significantly reduced the inhibitory effect of AA-5-HT (15 mg/kg, IP) on motility (Figure 2). In absence of AA-5-HT, I-RTX or SR144528 did not affect motility (geometric center: control: 4.5 ± 0.6, I-RTX: 4.7 ± 1.1, SR144528: 4.8 ± 0.7, n = 6 for each experimental group, P > .2).Figure 2Effect of IP-injected N-arachidonoylserotonin (AA-5-HT, 15 mg/kg) on intestinal transit in mice pretreated (IP) with the CB1 receptor antagonist rimonabant (SR1, 0.1 mg/kg, IP) or the CB2 receptor antagonist SR144528 (SR2, 1 mg/kg, IP) or the vanilloid receptor antagonist 5′iodoresiniferatoxin (I-RTX, 0.75 mg/kg IP). Transit was expressed as the geometric center of the distribution of a fluorescent marker along the small intestine (see Materials and Methods section). Bars represent the mean ± SEM of 8–11 animals. *P < .05 vs control and #P < .05 vs AA-5-HT alone.View Large Image Figure ViewerDownload (PPT)The results concerning the experiments carried out on FAAH- and CB1 receptor-deficient mice are shown in Figure 3. AA-5-HT (15 mg/kg) significantly (P < .05) reduced motility in both FAAH+/+ (Figure 3A) and in CB1 receptor+/+ (Figure 3B) mice. However, AA-5-HT produced no significant effect both in FAAH- and in CB1 receptor-deficient mice. Compared with wild-type mice, FAAH- and CB1 receptor-deficient mice showed a slight trend toward decreased or increased motility, respectively (Figure 3).Figure 3Effect of the FAAH inhibitor N-arachidonoylserotonin (AA-5-HT, 15 mg/kg, IP) on intestinal transit in FAAH-deficient (FAAH-KO) (A) or in CB1-deficient (CB1-KO) (B) mice (as compared with the corresponding wild-type (WT) littermates). Transit was expressed as the geometric center of the distribution of a fluorescent marker along the small intestine (see Materials and Methods section). Each bar represents the mean ± SEM of 6–8 animals. *P < .05 vs corresponding control (WT mice).View Large Image Figure ViewerDownload (PPT)Figure 4 shows the effect of IP-injected oleamide (1–20 mg/kg), oleoylethanolamide (1–20 mg/kg), and PEA (1–20 mg/kg) on motility. These amides significantly reduced intestinal motility, the effect being significant starting from the 5 mg/kg (oleamide) or 10 mg/kg (PEA and oleoylethanolamide) doses. In the presence of rimonabant (0.1 mg/kg), a significant inhibitory effect was observed only for PEA, and a significant reversion was achieved only for oleamide (Figure 5). Moreover, a dose of AA-5-HT (5 mg/kg, IP) which per se did not affect significantly intestinal motility increased the inhibitory effect of both anandamide and PEA on motility (Figure 6A and 6B). However, AA-5-HT did not affect significantly the dose response curve to the opioid drug loperamide (percentage inhibition of motility: loperamide, 0.03 mg/kg 20% ± 5%; loperamide 0.03 + AA-5-HT, 30% ± 7%; loperamide 0.1 mg/kg, 34% ± 6%; loperamide 0.1 + AA-5-HT, 42% ± 6%; loperamide 0.3 mg/kg, 42% ± 6%; loperamide 0.3 + AA-5-HT, 48% ± 6%; loperamide 1 mg/kg, 55% ± 5%; loperamide 1 mg/kg + AA-5-HT, 59% ± 6%; loperamide 3 mg/kg, 69% ± 7%; loperamide 3 mg/kg + AA-5-HT, 66% ± 7%; n = 6–8 for each experimental group).Figure 4Inhibitory effect of IP-injected oleamide, oleoylethanolamide, and palmitoylethanolamide (PEA) on intestinal transit in mice. Transit was expressed as the geometric center of the distribution of a fluorescent marker along the small intestine (see Materials and Methods section). Each bar represents the mean ± SEM of 7–10 animals. *P < .05 and **P < .01 vs vehicle control.View Large Image Figure ViewerDownload (PPT)Figure 5Effect of IP-injected oleamide, oleoylethanolamide, and palmitoylethanolamide (PEA) (all these biologic amides were used at the dose of 10 mg/kg) alone or in the presence of the CB1 receptor antagonist rimonabant (SR1, 0.1 mg/kg, IP). Transit was expressed as the geometric center of the distribution of a fluorescent marker along the small intestine (see Materials and Methods section). Bars represent the mean ± SEM of 7–11 animals. *P < .05 vs control and #P < .05 vs AA-5-HT alone.View Large Image Figure ViewerDownload (PPT)Figure 6Dose-related inhibition of intestinal motility by anandamide (A) or palmitoylethanolamide (PEA) (B) alone or in animals treated with the FAAH inhibitor AA-5-HT (5 mg/kg, IP). Bars represent the mean ± SEM of 6–8 animals. **P < .01 vs anandamide (or PEA) dose-response curve (statistical significance between 2 dose-effect curves).View Large Image Figure ViewerDownload (PPT)Endocannabinoid and Palmitoylethanolamide Content in the Small IntestineTable 1 shows that anandamide, 2-AG, and PEA were increased in the small intestine of animals treated with AA-5-HT (1–15 mg/kg, IP). AA-5-HT significantly increased anandamide and 2-AG levels starting from the 10 mg/kg dose, whereas PEA levels were increased only at the highest dose of AA-5-HT tested (15 mg/kg). By contrast, small intestines from FAAH-deficient mice revealed significantly increased levels of anandamide (but not 2-AG or PEA) as compared with intestines from FAAH+/+ mice (Table 2). The effect of oleoylethanolamide and oleamide on the intestinal levels of endocannabinoids and PEA is reported in Table 3. Both amides significantly increased the intestinal level of anandamide and reduced the level of 2-AG. PEA levels did not change after administration of either oleoylethanolamide or oleamide.Table 1Levels of Anandamide, 2-Arachidonylglycerol, and Palmitoylethanolamide in Mouse Small Intestine in Control Mice and in Mice Treated With the FAAH Inhibitor ArachidonoylserotoninControlAA-5-HT (IP)1 mg/kg5 mg/kg10 mg/kg15 mg/kgAnandamide (pmol/mg lipid)1.08 ± 0.01.02 ± 0.151.00 ± 0.051.85 ± 0.20aP < .05 vs control.1.55 ± 0.17aP < .05 vs control.2-AG (nmol/mg lipid)1.23 ± 0.201.30 ± 0.301.70 ± 0.202.30 ± 0.10aP < .05 vs control.2.15 ± 0.24aP < .05 vs control.PEA (pmol/mg lipid)6.60 ± 0.736.09 ± 0.706.11 ± 0.745.70 ± 0.459.36 ± 0.62aP < .05 vs control.NOTE. Results are expressed as the mean ± SEM from 4 animals.2-AG, 2-arachidonylglycerol; PEA, palmitoylethanolamide.a P < .05 vs control. Open table in a new tab Table 2Levels of Anandamide, 2-Arachidonylglycerol, and Palmitoylethanolamide in Mouse Small Intestine of Wild-Type or FAAH-Deficient MiceWild-type miceFAAH-deficient miceAnandamide (pmol/mg lipid)1.30 ± 0.333.61 ± 0.66aP < .05 vs wild-type mice.2-AG (nmol/mg lipid)1.34 ± 0.372.15 ±" @default.
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• W2093229075 title "Fatty Acid Amide Hydrolase Controls Mouse Intestinal Motility In Vivo" @default.
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