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Pain 139 (2008) 190–200

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Endocannabinoid and serotonergic systems are needed
for acetaminophen-induced analgesia
Christophe Mallet a,b, Laurence Daulhac a,c, Jérôme Bonnefont a,b, Catherine Ledent d,
Monique Etienne a,b, Eric Chapuy a,b, Frédéric Libert a,b,e, Alain Eschalier a,b,e,*
a
INSERM E9904, U766, Facultés de Médecine et de Pharmacie, 28, Place Henri Dunant, B.P. 38, F-63001 Clermont-Ferrand Cedex 01, France
b
Clermont Université, Université d’Auvergne, Faculté de Médecine, Laboratoire de Pharmacologie Médicale, F-63001 Clermont-Ferrand, France
c
Clermont Université, Université d’Auvergne, Faculté de Pharmacie, Laboratoire de Pharmacologie, F-63001 Clermont-Ferrand, France
d
IRIBHM, Université Libre de Bruxelles, B-1070 Bruxelles, Belgium
e
CHU Clermont-Ferrand, Service de Pharmacologie, Hôpital G. Montpied, F-63003 Clermont-Ferrand, France

Received 16 October 2007; received in revised form 18 January 2008; accepted 24 March 2008

Abstract

Acetaminophen is the most used analgesic/antipyretic drug. Its unclear mechanism of action could rely on cyclooxygenase inhi-
bition, NO synthesis blockade or reinforcement of the serotonergic system. Here we show that in thermal, mechanical and chemical
pain tests, AM-251, a specific CB1 receptor antagonist, abolished the analgesic action of acetaminophen, which was also lost in CB1
receptor knockout mice. Moreover, acetaminophen was shown unable to bind to CB1 receptors demonstrating an indirect involve-
ment of these receptors in the analgesic effect of this compound. Accordingly with these results, we also demonstrated that the inhi-
bition of FAAH, an enzyme involved in the cerebral metabolism of acetaminophen into AM404, known to reinforce the activity of
the endocannabinoid system, suppressed the antinociceptive effect of acetaminophen. In addition, similarly to the interaction of
acetaminophen with bulbospinal serotonergic pathways and spinal serotonin receptors, we observed that the antinociceptive activity
of ACEA, a CB1 receptor agonist, was inhibited by lesion of bulbospinal serotonergic pathways and antagonists of spinal 5-HT
receptors. We therefore propose that acetaminophen-induced analgesia could involve the following sequence: (1) FAAH-dependent
metabolism of acetaminophen into AM404; (2) indirect involvement of CB1 receptors by this metabolite; (3) endocannabinoid-
dependent reinforcement of the serotonergic bulbospinal pathways, and (4) involvement of spinal pain-suppressing serotonergic
receptors.
Ó 2008 Published by Elsevier B.V. on behalf of International Association for the Study of Pain.

Keywords: Acetaminophen; Endocannabinoids; Fatty acid amide hydrolase; Nociception; Serotonin

1. Introduction ity of the descending serotonergic pathways on spinal
nociceptive processing, which has been supported by dif-
The mechanism of the antinociception elicited by ferent groups [5,43,48]. Indeed, the lesion of the bulbo-
acetaminophen is not yet elucidated. One main hypo- spinal descending serotonergic pathways abolishes the
thesis relies on the reinforcement of the inhibitory activ- antinociceptive action of acetaminophen [48]. More-
over, our group demonstrated that different spinal 5-
HT receptor subtypes are involved in the antinociceptive
*
Corresponding author. Address: INSERM E9904, U766, Facul- effect of acetaminophen in rats [6,39] and recently con-
tés de Médecine et de Pharmacie, 28, Place Henri Dunant, B.P. 38,
firmed this serotonergic mechanism in humans [42].
F-63001 Clermont-Ferrand Cedex 01, France. Tel.: +33 4 73 17 82 32;
fax: +33 4 73 27 71 62. However, those mechanisms or others do not explain
E-mail address: alain.eschalier@u-clermont1.fr (A. Eschalier). how the action of acetaminophen is initiated.

0304-3959/$34.00 Ó 2008 Published by Elsevier B.V. on behalf of International Association for the Study of Pain.
doi:10.1016/j.pain.2008.03.030

respectively.4. 5. duration of the response in seconds during the two typical phases of nociceptive behavior (phase I: 0–5 min. the combination of D9-THC and pelvic girdle and a 25-gauge  1-inch needle connected to a cannabidiol is proposed in the treatment of pain for 25 ll Hamilton syringe was inserted into the subarachnoidal patients with multiple sclerosis [46]. mice [28] and wild-type mice (CD1 background) were gener. a CB1 recep- tor agonist to compare it with that demonstrated for 2. Animal care and experiments hind paw of the animals until a squeak (vocalization threshold) were carried out according to the guidelines of the Committee was obtained (cut-off: 750 g). 2. The syringe was held in position for few seconds periaqueductal gray (PAG) or the rostroventral medulla after the injection of a volume of 10 ll/rat. 55% humidity) and kept under a 12/12 h light/dark cycle. lase (FAAH). respectively. mainly metabolized by the fatty acid amide hydro- ing the experiments in order to acclimatize.1. result has to be cautiously interpreted. were housed under controlled environmental conditions (21– glycerol.7-Dihydroxytryptamine (5.000g for 15 min. injected subcutaneously (s. induce antinociceptive effects [24. activation of this system by exogenous ligands for cannabinoid (particularly CB1) receptors induces antin. 10 min for ACEA in rats). Intrathecal (i.6 mg/l.1 M phosphate buffer (pH 4.33] but also in several animal models of chronic tre et al. [34]. lumbar dorsal horn samples were homogenized in a saturated reinforcement of the serotonergic system.2. C. Drugs were administered at different devoid of any direct effect on CB1 receptors. 2% maintenance) according to Mes- [16. and the monoacylglycerol lipase.) into the dorsal surface of the Our results demonstrated that acetaminophen. Animals 2. (ii) we determined if this involvement was direct microliters of this buffer was injected in the HPLC system with or not.7-DHT the endocannabinoid system could be on the basis of the efficacy by determining 5-HT lumbar levels by HPLC. tutional Ethic Committee for animal experiments. Desipramine compound able to inhibit the reuptake of anandamide (10 mg/kg) was i. diluted in a pH 11 gly- cannabinoid system by performing three experimental cin buffer) and extracted with 2. Male CB1 null mutant (Cb1/) weight: 30 g). / Pain 139 (2008) 190–200 191 The discovery of an involvement of endocannabi. of the injected paw was monitored by measuring the total ment of the activity of the 5-HT bulbospinal pathways. were measured by applying an increasing pressure to the right ously supplied by Dr Ledent. We therefore 45. Animals spectives [12. Mallet et al. until a tail flick reported that cerebral injection of cannabinoids in the was elicited.2. On activity of acetaminophen may rely on an interaction the day of the experiment.4. a intrathecally 7 days before the experiment.24. Anandamide and 2-arachidonoyl. The In this line. purchased from Charles River.33]. for Research and Ethical Issues of IASP [54] and to our Insti- noids on pain modulation opens new mechanistic per. 60.32.) injections were performed under isoflurane ociception in various acute pain tests in rodents anesthesia (4% induction.51]. Lesion of the descending serotonergic pathways modulation of descending pathways to inhibit pain pro- cessing at the spinal level [30.4. The organic layer was back-extracted with receptors on the acetaminophen-elicited antinocicep. KCl solution and centrifuged at 15.t. hind paw. dissolved Interestingly. Several studies space between lumbar vertebrae L5 and L6. we investigated the possible interaction resulting supernatants were mixed with 400 ll of an internal between acetaminophen-induced antinoception and the standard (n-methyl-serotonin 0. The anesthetized rat was held in one hand by the pain [17]. Briefly. 90 and 120 min after administration) before sacrificing hypothesized that the interaction of acetaminophen with them and removing spinal cords in order to confirm 5.44]. Treatments were done after the . therefore suggesting the 2. Formalin test Rats and mice received 50 and 25 ll of 2. 100 lg/rat). Biting and licking antinociceptive effect that could lead to the reinforce.5 ml of dichloromethane/n- series: (i) we first studied the involvement of CB1 butanol (75/25). in saline containing 0. serotonergic descending bulbospinal pathways and spinal 5-HT receptors in the antinociceptive effect of 2. with food and water ad libitum for a week prior to start- tors. injected 30 min before 5. Paw pressure test Rats were submitted to the paw pressure test using an Ugo Adult male Sprague–Dawley rats weighing 175–200 g were Basile analgesimeter (Apelex. expressed in grams (g).p.7-DHT. two endogenous ligands of CB1 and CB2 recep. Methods 20–40 min for rats or 15–40 min for mice).4). and (iii) we assessed the involvement of the electrochemical detector. Intrathecal injections larly. (RVM) elicits antinociception. phase II: 2. tip diameter of the probe: 1 mm. One-hundred tion.2 mg/ml ascorbic acid.7-DHT by catecholaminergic neurons. Simi- 2.5% formalin acetaminophen.3. even if this last drug and submitted to the paw pressure test (before and 15. 300 ll of 0. Moreover.1. needed a times before formalin (40 min in rats or 30 min in mice for FAAH-dependent metabolism to exert its CB1-mediated acetaminophen. recent findings showed that acetamino. animals were treated with the active with the endocannabinoid system [37].c. 22 °C. was administered phen could be metabolized in the brain into AM404. Behavioral pain tests arachidonyl-20 -chloroethylamide (ACEA). Nociceptive thresholds.7-DHT to prevent [14] thanks to FAAH [23] and that the antinociceptive the reuptake of 5. 30.

the following groups were conducted. treatments were administered. (WAY-100. different animals were used in each The general behavioral tests performed to screen for typical experiments (n = 6–10 per treatment. assessed by the total number of pixels that changed color between 2 successive pictures (from black to white and from 2. four groups of different animals were Antinociception was assessed using the paw pressure test injected as follows: vehicle + vehicle. URB597. 30. 3–4 doses for each) or vehicle. 5. namely. 90 and 120 min after drug administration. when an animal changed its position). N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]- remained motionless on the ring was recorded. Antinociception inhibitors was tested. 60 and 120 min post.3 °C. tropisetron (0.) and PMSF (10 mg/kg) subcutaneously 25  25  40 cm enclosures. mometer (Ellab. AM-251 thermistor probe (Ellab.7. baseline carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) was temperatures were taken every 30 min for 90 min using a from Cayman Chemical (France). while the other drugs and inserted approximately 7 cm into the colon. were dissolved in sterile physiological serum (0.5. The test was ethyl]-N-2-pyridinylcyclohexane-carboxamide maleate salt conducted for 4 min at 15. Mallet et al. pramine (10 mg/kg) and AM-251 (3 mg/kg) were administered France) comprised a digital video camera set above 4 dark intraperitoneally (i. 60 Evaluation of the affinity of acetaminophen for the CB1 and 120 min post-administration. treatments in the different blocks were randomized. Data from forma- perature was recorded 15. the order of treatments being different from one 2.2.5.4. Catalepsy to the protocol of Rinaldi-Carmona et al. ViewPoint. Cookson (UK). 30. according 2.9. Data are presented as the means ± SEM. desi- rats (VideoTrack. A computer was used to analyze (s.). vehicle + active drug described above. which was lubricated and ACEA were dissolved in DMSO. Acetaminophen (100– 2. 2.) while ACEA (1– The system used for recording spontaneous activity of the 10 mg/kg). The ani. Statistical analysis only the tail being held gently between two fingers. desipramine. (acetaminophen or ACEA).5 lg/rat) and digital pictures (25 frames/second). ments) which were performed in a quiet room. injected with active drug (acetaminophen or ACEA. and tropisetron from Novartis (France). Denmark). Drugs of a plastic ring (12 cm diameter) fixed horizontally at a height allowing the hindpaws of rats to just touch the bench. The apparatus consisted 2.8.o. 60 and 120 min post. PMSF. Following the baseline interval. 90 and 120 min after drug ment. URB597 (0. and 60 min.7-DHT). CB1 receptor binding assay white to black. 60.192 C. [45]. One injury.5. Treatments were done after the measurement of two block includes a number of animals corresponding to the num- consecutive stable withdrawal latency values and testing was ber of the different treatments administered in each experi- performed 15.1.c. all animals in a same block were tested in the same short laps of time. Denmark) was used to record body tempera- ture. catalepsy and hypothermia [8] were dose–effect relationship was studied.7-dihydroxytryptamine creatinine sulfate administration. All experiments were performed blind by a single experi- menter using a parallel group design.6.9% NaCl). 30.5. Sponta- neous locomotor activity was counted for 5 min at 15.7-DHT (100 lg/rat) were administered intrathecally. 30 - perature reading. Rats were unrestrained throughout the experiment. Body temperature N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2. A digital ther. Treatments were ran- 2. respectively) were analyzed by a one-way ANOVA .15 mg/kg) haloperidol (5 mg/kg).4. the number of blocks corresponds to the number of administration. haloperidol and Phenylmethylsul- fonyl Fluoride (PMSF) were purchased from Sigma (France). Body tem. antinociception. The rat Acetaminophen was generously provided by Bristol-Myers- was placed across the ring and the time (s) during which the rat Squibb (France). hypothermia and locomotor activity assessment (at 2 h administration. animals per treatment. antagonist or inhibitor + active drug. lin test. Tail immersion test domized and administered according to the method of equal Tail of the rats was immersed in a hot water bath main.4-dichlorophenyl)-4- Rats were placed individually into an environmental room methyl-1H-pyrazole-3-carboxamide (AM-251) and Arachido- maintained at a constant temperature of 21 ± 0. Assessment of tetrad effect block to an other. Experimental procedure values and testing was performed 15. antagonist or inhibitor + vehicle. Prior to drug administration. Catalepsy was measured using the ‘‘ring test” described by Pertwee [41] modified for the rat [9].p. with 2. blocks in order to assess the effect of the different treatments tained at 46 °C.3.5. WAY-100635 (40 lg/rat). according to the experi- high dosage cannabinoid effects. (5. 60. 30. cannabinoid receptor in transfected CHO cells was performed in a radioligand binding assay using [H3]CP 55940. / Pain 139 (2008) 190–200 measurement of two consecutive stable vocalization threshold 2. nyl-20 -chloroethylamide (ACEA) were obtained from Tocris mals were allowed to acclimate for 60 min before the first tem. 45. Spontaneous locomotor activity 600 mg/kg) was administered orally (p. 45. Champagne au Mont d’Or. 30. When the motor impairment. The time latency for tail withdrawal was then (blindly administered) at the same time interval to avoid determined and a cut-off time of 30 s was applied to avoid unverifiable and time-variable environmental influences.635).3. When the influence of antagonists or 2. Animal activity was 5.

Results 3. Interaction between acetaminophen and CB1 fied with acetaminophen. an irreversible brain-penetrating FAAH inhib- threshold increase: 97. using 5.3. when the F-value tions as high as 1 mM (percentage of specific binding: was significant.1. 2d). We first performed a dose–response effect of ACEA 3.2% inhibition in phase I and II. Acetaminophen/endocannabinoid system relationship We assessed the influence of FAAH inhibitors on the antinociceptive activity of acetaminophen. a 5-HT3/4 receptor 3. 3a and b). Locomotor depressive and hypothermic effects respectively) and led to the suppression of the effect of induced by acetaminophen (300 mg/kg. These results indicated that acetaminophen icance was set at p < 0. mediated by CB1 receptors as a pre-treatment with To further compare the serotonergic profile of acet- AM-251 (3 mg/kg. respec- the F-value was significant.2 ± 3. (30. 1c) aminophen (Fig. 1b) and tail immer. 6. while haloperidol.) in order to look at the antinociceptive activity of this Those results suggested the involvement of CB1 CB1 agonist in the paw pressure test and compare its receptors in the antinociceptive effect of acetaminophen. aminophen in the formalin test and the paw pressure ing of [H3]CP 55940 by acetaminophen at concentra.5 ± 0.9 and dependently induced three of these effects: we observed 2.o. URB597 (0. a selective CB1 receptor 3. as well as a reduction in locomotor by AM-251. Evidence of a CB1 receptor involvement in the (10 mg/kg. 4c and d). i. Fig.3 ± 10.8 mg/kg for acetaminophen and ACEA.6. a two-way ANOVA analysis was performed and. tail immersion and tetrad 8. a dose-dependent antinociceptive activity from 100 to at the peak effect time.635 and tropisetron.1 ± 10. activity and a hypothermia which were delayed in com. Acetaminophen-induced antinociception relies on FAAH-dependent AM404 formation 3. However. even at 600 mg/ the 5-HT levels in the dorsal horn of the spinal cord kg. For paw pressure. a Dunnett’s test was used to ana. reversed the antinociceptive effect of acet- We observed no significant displacement of the bind.2. ACEA induced a dose- We therefore looked at the ability of acetaminophen to dependent antinociceptive effect for a maximal duration induce cannabinoid tetrad effects. i. s. 2b. test. a CB1 receptor antagonist (Fig. cord tissue for control and 5.2. also reversed the antinociception elicited by acet- sion (maximal latency time increase: 37 ± 6%.) failed to abolish these two effects aminophen and a CB1 receptor agonist. 2c).p. Fig.3% (2301 ± 114 vs 103 ± 7 ng/g of spinal active in our conditions (Fig. activates CB1 receptors via an indirect pathway. whether ACEA-elicited antinociception was involving the same spinal serotonergic receptors than those identi- 3. In order to demonstrate that the involvement of CB1 tor knockout mice. ED50s were 125.0% for acetamino- tests.7-DHT. Mallet et al. PMSF 3. 2a).2%. significantly inhibited the antinociceptive effect of cant antinociceptive activity in the formalin.4 ± 14. via a serotonergic bulbospinal mechanism To confirm these observations.05.6 ± 1.o. We then examined the action of ACEA (3 mg/kg) parison with the antinociceptive effect and needed higher after lesioning the serotonergic bulbospinal pathways doses (300–600 mg/kg. used at a dose shown to be able to abol- antinociceptive effect of acetaminophen ish the FAAH-dependent metabolism of acetaminophen Acetaminophen (300 mg/kg. we investigated (Fig. 5c).p. it did not produce any significant effect when spinal serotonergic pathways and spinal 5-HT receptors administered in Cb1/ mice (Fig. Fig. We observed that the . Fig. 5b). The level of statistical signif- receptor.1%) and phase II (37.p. [23]. 3. 4a and b). lin tests (Fig.1%. i. p. C. used as positive control.1. tests. Acetaminophen dose of 45 min for 10 mg/kg (Fig. paw pressure (maximal vocalization kg. p.9 ± 2. 1d).2.p. In each test.8% and 44.1.) induced a signifi. While acetaminophen reduced the receptors in the antinociceptive effect of acetaminophen nociceptive behavior elicited by formalin in phase I was in line with its previously described serotonergic (29.2 ± acetaminophen in both the paw pressure and the forma- 7. respectively [1.) were not ACEA in the paw pressure test (Fig.c). CB1 receptor agonist. 1a). we examined the antinociceptive activity of acetaminophen in CB1 recep. We previously showed that receptors intrathecal injection of the 5-HT1A receptor antagonist WAY-100.).5.2. acetami. potency with acetaminophen. In addition. itor. tively) in transfected CHO cells expressing CB1 lyze the time-course of the effects. Such a treatment significantly reduced nophen did not induce any catalepsy. respectively.40].1.).8% and 11. elicits antinociception antagonist. 105 and 103 M. mechanism. this effect was completely abolished by AM-251 (3 mg/kg.1 ± 6. ACEA. when phen concentrations of 107. 5a). / Pain 139 (2008) 190–200 193 followed by a Student–Newman–Keuls’ test. Tetrad effects of acetaminophen (i. Acetaminophen does not bind to CB1 receptors antagonist. was by 95. we studied the involvement of both bulbo- type mice.1%) in wild. The effect of ACEA was inhibited 300 mg/kg (Fig.1.7-DHT-treated rats.2. in the effect of a CB1 receptor agonist.15 mg/ respectively.

/ .o. i.) .) (i .t.Ace Aceta ** 400 * 300 200 0 0 15 30 45 60 75 90 105 120 Time (min) 11 Veh h .Veh AM-251 . 6b). + + 600 ** Veh h . + .o.o. The effect of acetaminophen (Aceta. . 1.) . + Aceta a (p. + .) ./ - Fig. .) . AM-251 was administered 10 min before acetaminophen. **p < 0. + WT C b1 . (d) Formalin test was performed in wild-type (WT) or in Cb1/ mice after treatment by acetaminophen (Aceta. i.V eh ** ** ** Veh h .194 C. p.01 as compared with the vehicle-treated group.V eh ** Veh h . Mallet et al.) . by tropisetron (0.05.Veh AM -251 . administration of AM-251 (3 mg/kg) or vehicle. The CB1 receptors are involved in the antinociceptive effect of acetaminophen. Error bars represent SEM. WT C b1 . In all tests.p.) . + AM251 (i. + + AM251 (i. and (c) the tail immersion test in rats after i. p.) was assessed (a) in the formalin. 300 mg/kg. *p < 0.Aceta Time withdrawal latency (s) 10 AM-251 .635 (40 lg/rat.) or vehicle.5 lg/rat.) (i . + . n = 6–8 per group. .p. + .Ace Aceta 9 8 7 6 0 0 15 30 45 60 75 90 105 120 Time (min) Phase I Phase II 160 400 Biting & licking time (s) Biting & licking time (s) 140 350 120 * 300 100 250 * 80 200 60 150 40 100 20 50 0 0 Acet a (p.) in the formalin test (Fig.t.Aceta Vocalization Threshold (g) 500 AM -251 . (b) the paw pressure. antinociceptive activity of ACEA was blocked by WAY.o. / Pain 139 (2008) 190–200 Phase I Phase II 135 225 Biting & licking time (s) Biting & licking time (s) 120 200 105 175 90 * 150 75 125 ** 60 100 45 75 30 50 15 25 0 0 Aceta a (p. + Aceta (p.o.p. 300 mg/kg.) in the paw pressure test 100.o. 6a) and (Fig.

from the inhibition of cyclooxygenases [36. Several hypotheses have been emitted going.2 Aceta 100 mg/kg 150 * Aceta 200 mg/kg 36. In all tests. n = 6–8 per group. i. n = 6–8 per group. **p < 0. 300 or 600 mg/kg.4 300 Temperature (°C) 38. AM-251 was administered 10 min before acetaminophen.) or vehicle. Error bars represent SEM.001 as compared with the same treated group. 100.8 3 36.4 2 36. events that could serve as a new basis to resolve this pound.) or vehicle (a) in the paw pressure test. p. 2.0 Aceta 300 mg/kg 50 Aceta 600 mg/kg * 0 0 0 15 30 45 60 75 90 105 120 0 15 30 45 60 75 90 105 120 Time post dose (min) Time post dose (min) Fig.05. Data shown were obtained 2 h after administration of acetaminophen. to the From its initiating molecular mechanism to the reinforcement of the serotonergic system [5.8 Veh * Aceta 300 mg/kg Aceta 100 mg/kg * 36.) or vehicle and acetaminophen (300 mg/kg.0 * 7 * Temperature (ºC) 6 * * 37. mediators/receptors responsible for antinociception. as compared with the vehicle-treated group. (c) in the ring test and (d) for hypothermia. 4.8 After treatments 38. Mallet et al. 3. . The CB1 receptors are not involved in the motor impairment and hypothermia due to acetaminophen. Data shown were obtained 1 h after administration of acetaminophen. Discussion for instance.8 38. Animals were tested after administration (at time 0) of acetaminophen (aceta.0 1 0 0 Veh Veh AM-251 AM-251 Veh Veh AM-251 AM-251 Treatments: Treatments: Veh Aceta Veh Aceta Veh Aceta Veh Aceta Fig.4 100 Aceta 600 mg/kg Aceta 200 mg/kg Haloperidol5 mg/kg 36. *p < 0. C.48]. before treatments (a) or with the vehicle-treated group (b). we report than one century after the discovery of this com. They all failed to fully explain the action of the drug. (b) by video tracking.6 ** 5 37.4 8 Locomotor activity (*100 cm) 38. to the inhibition of NO synthesis [4].6 200 Latency( s) * Veh 37. injection of AM-251 (3 mg/kg) or vehicle and an oral administration of acetaminophen (300 mg/kg) or vehicle. (a) Rectal temperature was measured in rats before and after treatments with an i.) was used as a positive control for catalepsy (c). (b) Motor activity was assessed in rats treated with AM-251 (3 mg/kg.o. Dose–effect relationship of acetaminophen in the behavioral tetrad effect. Before treatments 38.42. mystery. *p < 0. Here.p. the singular mechanism of action of acetaminophen probably because some gaps remain to be filled remains elusive and is still a matter of debate more between all those different systems. i. Haloperidol (5 mg/kg.01. p.p.p. Error bars represent SEM.43.2 *** 4 36. 200. ***p < 0.38]. / Pain 139 (2008) 190–200 195 Antinociception Motor impairment 600 * Veh Aceta 100 mg/kg * Veh 14 Vocalization Threshold (g) Aceta 200 mg/kg Locomotor activity (*100 cm) 500 Aceta 100 mg/kg 12 Aceta 300 mg/kg * Aceta 200 mg/kg Aceta 600 mg/kg * * Aceta 300 mg/kg 10 400 * 8 6 300 * 4 * 200 2 * 0 0 0 15 30 45 60 75 90 105 120 0 15 30 45 60 75 90 105 120 Time post dose (min) Time post dose (min) Hypothermia Catalepsy 38.0 * * 250 * 37.05.o.

196 C. block the antinociceptive effect of acetaminophen.o. a specific antagonist of would be a major component of the activity of acetami. Error bars represent SEM. If our results are in accordance with a recent the different hypotheses. p. Firstly.) . + Aceta (p.) was assessed in the formalin (a and c) and the paw pressure tests (b and d) after administration of PMSF (10 mg/kg. .01 as compared with the vehicle-treated group. + PMSF (s. / Pain 139 (2008) 190–200 Phase I Phase II 135 250 Biting & licking time (s) Biting & licking time (s) 120 225 105 200 90 * 175 75 150 * 125 60 100 45 75 30 50 15 25 0 0 Aceta (p.) .o. + + 700 ** Veh . + . p.) . 4. + .o. Our results propose that the endocannabinoid system acetaminophen and AM-251. s. We demonstrated the involvement of only using high doses of acetaminophen (up to CB1 receptors using both knockout mice insensitive to 1000 mg/kg.Veh Vocalization Threshold (g) Veh . in endocannabinoid system in different behavioral tests in this previous study. + + PMSF (s. .p. *p < 0.Aceta 500 400 300 200 -15 0 15 30 45 60 75 90 105 120 Time (min) Phase I Phase II 135 250 Biting & licking time (s) Biting & licking time (s) 120 225 105 200 * 175 90 75 150 ** 125 60 100 45 75 30 50 15 25 0 0 Aceta (p. In all tests.p. respectively.Veh Vocalization Threshold (g) 600 ** Veh .Veh 500 URB597 . + Aceta (p.15 mg/kg. + + 700 ** Veh .c. n = 6–8 per group. The effect of acetaminophen (Aceta.) or vehicle (a and b) and URB597 (0.) .) .Aceta 600 ** PMSF . . CB1 receptors.p.) or vehicle (c and d). i. + URB597 (i.c. we observed that the report [37].) . which inhibited the effect of acetamino- nophen and could be an essential link between some of phen. Indeed. the effect of the drug was assessed rats and mice.) in the thermal hot plate nociceptive .Aceta URB597 .Veh PMSF . PMSF and URB597.o. **p < 0.05. PMSF and URB597 were administered 20 and 10 min before acetaminophen. + + URB597 (i.) . .c. + . Mallet et al.o. The FAAH inhibitors.Aceta ** 400 300 200 -15 0 15 30 45 60 75 90 105 120 Time (min) Fig.) . + . it is noteworthy that this hypothesis needed antinociceptive activity of acetaminophen relied on the to be verified using different noxious stimuli. 300 mg/ kg.o.

) or vehicle in the paw pressure test. we used low FAAH-inhibiting doses of URB597 (0. which induce a false antinociception when Kathuria et al.Veh 500 AM . the results obtained here in pain tests dem- 0 15 30 45 60 75 90 105 120 onstrated that intact CB1 receptors are needed for the Time (min) antinociceptive effect of acetaminophen. / Pain 139 (2008) 190–200 197 600 * * ify this last point. [25] and Compton and Martin [7].DHT . to mention that the inhibition of FAAH can reduce lesioned serotonergic bulbospinal pathways by 5.7-DHT. Further work is needed to ver. observed in the ciception in our conditions.) that did not induce any intrinsic antino- motor impairment and hypothermia. More. 5. (b) those data. However.ACEA metic compound.) was then assessed in the paw the antinociceptive activity of acetaminophen to assess pressure tests 10 min after i.47]. and diazepam) can induce some of the tetrad effects showing the lack of 200 specificity of this procedure [53]. by the analgesic drug. over. but failed to observe catalepsy. Using orally administered doses of acetaminophen from 300 100 to 600 mg/kg. [22] failed to show any inhibitory we showed. Those doses can induce toxicity [26]. motor reflexes and thermal stimulus are used [29]. which could represent SEM. as previously shown by present work. 0 Whatever. which can be further transformed into AM404 in the brain after a FAAH- 300 dependent conjugation with arachidonic acid [23]. desipramine. In line with of ACEA (1.DHT . i. A recent study suggested that every- ** thing could start from the liver where acetaminophen is 400 metabolized into p-aminophenol. in these conditions. Secondly. ethanol.p. s. The question would now be how CB1 receptors are activated.p.c.Veh *** *** Veh . (c) Vocalization thresholds were determined in rats after i. we 600 could suspect that acetaminophen is not a cannabimi- Veh . we studied the effect of FAAH inhibitors on The effect of ACEA (3 mg/kg.p. formation of AM404 seems to be necessary for acetami- ences in CB1 involvement due to the stimulus used or nophen-induced antinociceptive effect and could to the site where the noxious stimulus was applied account for an indirect involvement of CB1 receptors (somatic versus visceral).ACEA thermic effects by a CB1 receptor antagonist and is in 400 line with the finding that non-cannabinoid compounds (amphetamine.05. (a) Animals were tested after administration nabinoid-dependent antinociception [21. motor impairment. i.p. *p < 0. for the first time. Mallet et al. we observed three of the four tetrad effects (antinociception.52] or several cannabinoid receptor agonists [17]. [31] and Fride and Mechoulam [18] who 0 15 30 45 60 75 90 105 120 consider that only an activity of compounds in all four Time (min) tests would characterize them as cannabimimetics. Indeed.p. i.01. 300 pimozide. injection of ACEA (3 mg/kg) or vehicle in healthy rats or in rats with first. Serotonergic bulbospinal pathways are needed for CB1 agonist. 3. We want. This conclusion is reinforced by the lack of inhibition of its locomotor depressive and hypo- Vocalization Threshold (g) AM .19] and the reduction of degrada- Time (min) tion (by inhibiting FAAH) [20] of anandamide. n = 6–8 per group. pentobarbital. Error bars the degradation of anandamide [20]. we looked at the effect of acetaminophen on the classical ‘‘tetrad” which has Veh allowed to establish a cannabimimetic behavioral profile Vocalization Threshold (g) 500 ACEA 1 mg/kg * ACEA 3 mg/kg for the naturally occurring D9-THC and anandamide * ACEA 10 mg/kg 400 * [8. Thus FAAH-dependent induced abdominal writhes which could suggest differ.7 . injection of AM-251 (3 mg/kg) or the involvement of this metabolic pathway. that this enzyme is needed effect of a CB1 antagonist using phenylbenzoquinone. **p < 0.) and PMSF test. Haller et al. vehicle.251 . to avoid such an effect. Several Fig. ***p < 0. 10 mg/kg. morphine. scopolamine. 600 Veh .ACEA of Fowler et al. According to Mar- 0 tin et al.Veh ** Veh .ACEA we did not observe any binding of acetaminophen to CB1 receptors with a high affinity confirming the results VocalizationT hreshold (g) 5. hypother- 200 mia). studies also reported that AM404 could induce a can- induced antinociception.27]. as well as (10 mg/kg. C.001 as compared with account for an antinociceptive effect [25. [15]. further studies are .251 . Using these inhibitors. In order the vehicle-treated group.15 mg/kg.Veh 500 ** 5. 200 AM404 is in turn able to reinforce the activity of the 0 endocannabinoid system through both the inhibition 0 15 30 45 60 75 90 105 120 of cellular reuptake [3.7 . for the effect of acetaminophen. respectively.

6. n = 6–8 per group. This similar 5-HT-dependent mecha.) .40]. For instance. firmed in vivo.40] and that the source of spinal 5-HT the serotonergic system. inhibition during the acetaminophen-induced antinoci- tonergic system involved similar spinal 5-HT receptors ceptive and/or antipyretic activities will have to be con- to those involved in the antinociceptive action of acet. endocannabinoid system in pain modulation either at the demonstration that AM404 can be synthesized in spinal or supraspinal levels [35]. Notably.) .Veh 500 Tropisetron .p. + + WAY-100635 (i. + .5. Other studies stated that the antinociceptive activity of aminophen and 5-HT to produce antinociception.t. we demonstrated that the antinociceptive the tetrad tests [13]. the 5-HT1A recep. the actual modalities of this has been shown to depend on spinal serotonergic recep. .t. further studies rons which can reduce the GABAergic negative influ. ACEA-dependent recruitment of the sero.e.) .39. Spinal 5-HT receptors are involved in CB1 agonist-induced antinociception in rats. On the contrary. first reinforce the activity of the endocannabinoid sys- Different studies reported the implication of the tem and then that of the serotonergic one. needed to assess the antinociceptive effect and the mech. not found in blood after administration of acetamino- tor in the formalin test and the 5-HT3/4 receptor in the phen [23] might explain why acetaminophen does not paw pressure test. That would lead to suspect that acetaminophen would anism of action of AM404.p. + .t.11. 6. the stimula. Intrathecal administrations were performed 5 min before acetaminophen or saline. does not exert any significant anti-inflammatory effect.2. injection of (a) WAY-100. interaction and how this leads to the reinforcement of tors [6.ACEA Vocalization Threshold (g) Tropisetron . . whose stimulation has been shown to activity of the bulbospinal serotonergic pathways. i.50].ACEA 400 ** 300 200 0 0 15 30 45 60 75 90 105 120 Time (min) Fig. In addition. elicit antinociception in the PAG [30] and to be active in Accordingly. acetaminophen (or its metabolite AM404) with the Since the antinociceptive activity of acetaminophen endocannabinoid system.p.Veh ** ** Veh . The effect of ACEA (3 mg/kg.5 lg/rat) or vehicle.635 (40 lg/rat) or vehicle and (b) tropisetron (0. with a potency close to intact descending bulbospinal serotonergic pathways. However. comes exclusively from supraspinal centers and mainly It also remains possible that AM404 mediates the from RVM [35].01 as compared with the vehicle-treated group. exert any significant peripheral anti-inflammatory effect. + + 600 Veh .) was assessed (a) in the formalin and (b) the paw pressure tests after i. The involvement of a central COX Furthermore. + WAY-100635 (i. Error bars represent SEM. those of NSAIDs [23]. AM404 could activate the phen would occur through the reinforcement of the TRPV1 receptor. the fact that AM404 is aminophen and 5-HT [1. nism and the inhibition of the effect of acetaminophen This could therefore further explain why acetaminophen by inactivation of CB1 receptors suggest that the endo. i.) . the spinal cord after acetaminophen treatment and the tion of CB1 receptors in PAG and RVM reduced GABA presence of spinal CB1 receptors [23] might also suggest release from the presynaptic boutons of local interneu. cannabinoid system is an important link between acet. **p < 0. acetaminophen may rely on a decrease in spinal NO . mechanisms. an involvement of these receptors.198 C. Thus. Mallet et al. / Pain 139 (2008) 190–200 Phase I Phase II 150 350 Biting & licking time (s) Biting & licking time (s) 135 300 120 250 ** 105 ** 90 200 75 60 150 45 100 30 50 15 0 0 ACEA (i. will be needed to determine the site of the interaction of ence on the inhibitory descending pathways [49. AM404 has been shown effect induced by a CB1 receptor agonist (ACEA) needed to inhibit COX activities in vitro. we suspected that the participation of antinociceptive activity of acetaminophen through other the endocannabinoid system in the effect of acetamino. + ACEA (i.

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