![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Institut National de la Santé et de la Recherche Médicale/UdA E9904, Faculté de Médecine, Service de Pharmacologie, Centre Hospitalier Universitaire, Clermont-Ferrand, France (F.L., J.B., A.A., A.E.); Centre National de la Recherche Scientifique–Unité Propre de Recherche 1142 141, Montpellier, France (E.B., J.N.); and Institut National de la Santé et de la Recherche Médicale U288 Centre Hospitalier Universitaire Pitié-Salpêtrière, Faculté de Médecine, Paris, France (E.D., M.H.)
Received January 21, 2004; accepted June 11, 2004
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). Physicochemical, pharmacokinetic, and pharmacodynamic data are more in favor of a central effect of this molecule: acetaminophen 1) crosses the blood-brain barrier (Bannwarth et al., 1992; Courade et al., 2001a); 2) induces an antinociceptive effect after central administration (Gelgor et al., 1992; Alloui et al., 1996; Pélissier et al., 1996); and 3) inhibits responses in noninflammatory pain models after systemic administration (Hunskaar et al., 1985; Pélissier et al., 1996).
Various mechanisms have been evoked to explain the central action of acetaminophen, such as inhibition of central cyclooxygenases (Chandrasekharan et al., 2002) and/or inhibition of NO synthase (Ryu et al., 2000), but their contribution to its analgesic effect is a matter of debate (Mitchell et al., 1993; Amin et al., 1995; Farivar et al., 1996; Warner et al., 1999). Another evoked mechanism implicates the serotonergic system. Indeed, lesions of serotonergic pathways (Tjolsen et al., 1991) as well as inhibition of serotonin (5-HT, 5-hydroxytryptamine) synthesis by p-chlorophenylalanine (Pini et al., 1996) markedly reduced the antinociceptive effect of acetaminophen. On the other hand, acetaminophen treatment was shown to increase 5-HT levels in various central nervous system structures (Pini et al., 1996; Courade et al., 2001b) and reduce the density of cortical 5-HT2 receptors (Pini et al., 1996; Srikiatkhachorn et al., 1999). Even though both pro- and antinociceptive effects of serotonin can exist (for review, Millan, 2002), these data suggest that a serotonergic mechanism might be involved in the antinociceptive action of paracetamol.
In 1996, Pelissier et al. reported that the antinociceptive effect of systemically administered acetaminophen could be reversed by tropisetron, a 5-HT3 receptor antagonist. They suggested that the 5-HT3 receptor, which has been reported to have both pro and antinociceptive influences in the spinal cord (Bardin et al., 1997; Zeitz et al., 2002), could be involved in the antinociception mediated by acetaminophen. However, other data show that tropisetron is not a selective 5-HT3 receptor antagonist (Arranz et al., 1998; Maksay et al., 1999; Macor et al., 2001) and led us to reconsider the actual implication of 5-HT3 receptors in the antinociceptive action of acetaminophen. For this purpose, two series of experiments were performed: 1) in vitro electrophysiological recordings to assess whether acetaminophen directly activates 5-HT3 receptors and 2) in vivo pharmacological investigations aimed at assessing whether various 5-HT3 receptor antagonists and antisense oligodeoxynucleotides (AODNs) that down-regulate 5-HT3 receptor expression interfere with the antinociceptive action of acetaminophen.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
) was inserted in the vertebrate expression vector pcDNA3 (Invitrogen). For transient expression in stage V and VI Xenopus laevis oocytes, nuclear injection was performed as described previously (Bourinet et al., 1994) with 10 nl of the 5-HT3A encoding plasmid suspension. Oocytes were then incubated at 18°C for 2 to 4 days in ND96 medium on a rotating platform.
Electrophysiology. Macroscopic oocyte currents were recorded using two voltage-clamp electrodes with a GeneClamp500 amplifier (Axon Instruments, Union City, CA) in ND96 solution containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 2.5 mM sodium pyruvate, and 5 mM HEPES, pH adjusted to 7.45 with NaOH (Bourinet et al., 1994). Oocytes were placed in a small recording chamber continuously perfused by a gravity-driven perfusion system controlled by solenoid valves. The system allows a fast application of solutions. pClamp7 software (Axon Instruments) was used for data acquisition, and analysis was performed with pClamp8, Excel (Microsoft Corp. Redmond, WA), and Prism (GraphPad Software, San Diego, CA) software.
Three experiments were performed: a first based on the application of 10 µM 5-HT at different holding potentials to reveal the functionality of 5-HT3A receptors; a second with 5-HT and the 5-HT3 receptor antagonist, granisetron to confirm the involvement of these receptors; and a third to comparatively assess the effect of 5-HT, m-chlorophenylbiguanide (mCPBG), and acetaminophen on the 5-HT3 receptors. To be exhaustive, a last experiment was performed to determine whether acetaminophen could act as a 5-HT3 receptor antagonist. n = 5 to 32 according to the experiments.
In Vivo Studies
Animals. For all experiments, male Sprague-Dawley rats (180–200 g) were used (Charles River Laboratories, St Aubin les Elbeuf, France). Animals were divided into groups of six per cage and allowed to acclimate for 1 week at 21°C with free access to food and water.
Experimental protocols were implemented according to recommendations of the committee of research and ethics of the International Association for the Study of Pain, and submitted to the local ethical committee. Intrathecal (i.t.) injections were carried out according to the protocol described by Mestre et al. (1994).
Nociceptive Test (Paw Pressure Test). During all the experiments, the evaluation of antinociceptive effects was carried out using the paw pressure test (Randall and Selitto, 1957). Increasing pressures measured with an analgesimeter (tip diameter of the probe, 1 mm; weight, 30 g; Apelex; Ugo Basile, Comerio, Italy) were applied to the left hind paw of rats, with a cutoff at 750 g. Vocalization thresholds, considered nociceptive thresholds, were expressed in grams (baseline predrug vocalization thresholds range from 284 ± 28 g to 316 ± 25 g).
Treatment Protocol
Influence of 5-HT3 Receptor Antagonists on Acetaminophen-Induced Antinociceptive Effect. We studied the effect of tropisetron (0.5 µg/rat, i.t.), granisetron, and ondansetron (0.1, 1, 10, and 20 µg/rat, i.t.) on the antinociceptive activity of acetaminophen 400 mg/kg (p.o.). All the antagonists were injected 10 min before acetaminophen and were used at doses previously shown to inhibit the antinociceptive action of mCPBG in the same behavioral test in rat (Bardin et al., 1997). Vocalization thresholds were then measured every 15 min for 120 min (n = 8 per group) after administration of acetaminophen. Only one dose of tropisetron was used as a control experiment, because its ability to inhibit the effect of acetaminophen has previously been demonstrated (Alloui et al., 1996; Pélissier et al., 1996).
Antisense Experiments. To complete the results obtained with 5-HT3 receptor antagonists, an antisense approach was used, with AODNs designed to reduce 5-HT3 receptor synthesis. The base sequence of the 5-HT3 antisense was adapted from that of an already described antisense oligodeoxynucleotide sequence that efficiently down-regulates mouse 5-HT3 receptor expression (Paul et al., 2001): CGG GAT GCA GAG CGG CAT. The sequence of the negative control (mismatch) was made by inversion of the two consecutive highlighted bases at two points of the antisense sequence. A blast of the two sequences (http://www.ncbi.nlm.nih.gov/BLAST) enabled us to verify that oligodeoxynucleotides (ODNs) presented no other site of interaction.
Saline (10 µl/rat) and antisense or mismatch ODNs (12.5 µg/rat) were injected i.t. every 12 h for 4 days. Two active doses of mCPBG, determined by a dose-effect relationship study, were administered i.t. on the fifth day of the ODN injection protocol, and their antinociceptive effects were evaluated for 60 min. The same protocol was then applied to assess the antinociceptive activity of acetaminophen (400 mg/kg, p.o.) in rats treated with ODN.
All behavioral experiments were performed blind in a quiet room by a single experimenter using the method of equal blocks with randomization of treatments to avoid any uncontrollable environmental influence that might induce a modification in the behavioral response.
Validation of Antisense Efficiency by Immunoautoradiography. On the fifth day of antisense or mismatch ODN injections, the lumbar enlargement of the spinal cord was removed, and sections from fixed tissues were labeled using the protocol described by Doucet et al. (2000).
All immunolabeling experiments were performed using a purified rabbit polyclonal antibody directed against a 16-amino acid synthetic peptide corresponding to a selective portion of the second intracellular loop of the rat 5-HT3-A receptor subunit. These antibodies have been shown to specifically interact with the rat 5-HT3-A receptor subunit (Doucet et al., 2000). Coronal sections were successively incubated with anti-5-HT3A receptor antibodies, and 125I-IgG anti-rabbit IgG. Quantification of 125I-labeling of immunoautoradiograms was made using a Biocom image analyzer.
Drugs. Acetaminophen was from Bristol-Myers Squibb (Rueil-Malmaison, France). Tropisetron was from Sandoz (Rueil-Malmaison, France), and granisetron and ondansetron were from Glaxo-SmithKline (Marly-Le-Roy, France). mCPBG and serotonin (creatinine sulfate complex) were obtained from Sigma-Aldrich (l'Isle d'Abeau Chesne, France). ODNs were synthesized by Invitrogen (Cergy Pontoise, France). Products administered intrathecally were dissolved in sterile saline (0.9% NaCl; B Braun, Melsungen, Germany).
Expression of Results and Statistical Analyses. Results from electrophysiological and immunoautoradiographic studies are presented as the mean ± S.E.M. and compared using t test analysis. Results from behavioral studies are expressed as the difference (in grams) between vocalization thresholds after treatment and control predrug values. To investigate global effects, areas under the time course curves (AUC) of the antinociceptive effects were calculated using the trapezoidal method. Data were analyzed by a two-way analysis of variance followed, when the F-value was significant, by a Dunnett's test to study the time course of the effect or by a one-way analysis of variance to compare the effects of different treatments. Scores were considered significantly different when p < 0.05.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). Application of 10 µM 5-HT at different holding potentials revealed the typical functional characteristics of 5-HT3A channels with desensitization current kinetics at negative holding potentials (Fig. 1A) and a linear current-voltage profile with a reversal potential near 0 mV (Fig. 1B). Application of the specific antagonist granisetron (100 nM) blocked completely the 5-HT–evoked responses (data not shown). Homomeric 5-HT3A receptors were equally activated by 5-HT or by the specific agonist mCPBG as shown by the dose-response curves presented in Fig. 1C. In sharp contrast, application of acetaminophen (from 300 µM to 30 mM) was unable to evoke any 5-HT3A receptor activity (Fig. 1C). To confirm the lack of any influence of acetaminophen on 5-HT3A receptors, a potential antagonist activity of acetaminophen at 5-HT and mCPBG sites onto the 5-HT3A receptors was examined. No difference was observed between control and treated groups, indicating that acetaminophen has no 5-HT3 antagonist properties (Fig. 1D).
|
Evidence for a Lack of Involvement of 5-HT3 Receptors in the Antinociceptive Effect of Acetaminophen
Acetaminophen administered orally induced a significant and similar antinociceptive effect in all behavioral studies performed.
Influence of 5-HT3 Receptor Antagonists
Tropisetron (0.5 µg, i.t.) totally reversed the antinociceptive effect of acetaminophen. Groups treated with "saline + saline", "tropisetron + saline", or "tropisetron + acetaminophen" presented no significant differences, whereas acetaminophen induced a significant increase in vocalization thresholds compared with the control group (Fig. 2).
|
Neither ondansetron (Fig. 3, A and B) nor granisetron (Fig. 4, A and B), inactive on their own, significantly altered the antinociceptive effect of acetaminophen at doses up to 10 µg/rat. However, an important yet not significant decrease in AUC scores (–52 ± 20%) was observed after i.t. administration of 20 µg of granisetron. Significant differences were obtained between groups treated with "granisetron + saline" and "granistron + acetaminophen" at 40 and 60 min. For ondansetron, the resulting decrease in acetaminophen-induced antinociception was slighter.
|
|
Influence of 5-HT3A Receptor-Directed Antisense ODN
Immunoautoradiographic Labeling of Spinal 5-HT3 Receptors in AODN-Pretreated Rats. The labeling is especially intense within the superficial laminae of the dorsal horn, as expected from the known distribution of 5-HT3 receptors in the rat spinal cord (Laporte et al., 1992) (Fig. 5). Compared with saline-treated rats, AODN treatment significantly reduced the optical density in laminae I and II by 29.2 ± 7.9%, whereas mismatch ODN did not significantly change the immunoautoradiographic labeling (Table 1).
|
|
Antinociceptive Effect of mCPBG. mCPBG induced a dose-dependent antinociceptive effect that was significant for all the doses tested (Fig. 6). Both intensity (maximal increase, 188 ± 36 g) and duration (maximal, 60 min) of the effect were dose-dependent as confirmed by AUC analysis. The two highest doses (1 and 10 µg/rat, i.t.) were selected to study the influence of AODNs.
|
Pharmacological Validation of the Efficacy of AODNs. As illustrated in Fig. 7A, it was verified that pretreatment with AODNs significantly attenuated the antinociceptive effect of mCPBG at the two i.t. doses tested. Indeed, AUC analysis showed that, at 1 and 10 µg, the effect of mCPBG was reduced by 58 ± 10% and 60 ± 2%, respectively, in AODN-pretreated rats. On the other hand, mismatch ODNs did not significantly inhibit the effect of mCPBG at 1 µg/rat, i.t. (Fig. 7A).
|
Effect of AODN Pretreatment on Acetaminophen-Induced Antinociceptive Effect. Pretreatment with ODNs induced no reduction in the antinociceptive effect of acetaminophen (400 mg/kg, p.o.). As illustrated in Fig. 7B, animals treated with acetaminophen had similar vocalization thresholds regardless of the pretreatment (saline, AODNs, or mismatch ODNs) they had received.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). In vitro experiments suggest that acetaminophen could inhibit COX-3, a putative new cyclooxygenase isoform more potently inhibited by NSAIDs that also inhibit COX-1 and COX-2 isoforms (Chandrasekharan et al., 2002) in opposition to acetaminophen. However, some reports suggest that either the acetaminophen concentrations needed to inhibit COX-3 are difficult to reach with an oral dose of 0.5 to 1 g (Schwab et al., 2003) or question the existence of the catalytically active form of COX-3 in humans (Dinchuk et al., 2003). The hypothesis of a serotonergic mechanism is based on in vivo experiments involving behavioral assessments (Tjolsen et al., 1991; Pini et al., 1996) and biochemical assays (Srikiatkhachorn et al., 1999; Courade et al., 2001b). In terms of receptors, the 5-HT3 receptors were suspected to be involved because of the inhibitory effect of tropisetron (Pélissier et al., 1996), but an interaction with other 5-HT receptors was also described previously (Courade et al., 2001c). Two main results were obtained in this work based on this central serotonergic hypothesis: 1) acetaminophen did not act directly on the 5-HT3A receptors and 2) its antinociceptive effect was inhibited neither by two 5-HT3 receptor antagonists nor by an AODN-induced down-regulation of a 5-HT3 receptor in the spinal cord.
Previous studies aimed at identifying possible binding sites for acetaminophen showed a lack of affinity of this drug for 5-HT3 or other 5-HT receptors (Pélissier et al., 1996; Raffa and Codd, 1996). However, these data were obtained for acetaminophen concentrations up to 10 µM, and one could not exclude some interaction with 5-HT3 receptors at higher concentrations of the drug. The results obtained in the present electrophysiological studies largely demonstrated the inability of high concentrations of acetaminophen (up to 30 mM) to act on recombinant 5-HT3A receptors. Therefore, these data exclude any direct agonist or antagonist effect of the analgesic drug on 5-HT3A receptors. Two 5-HT3 receptor subunits have been identified to date (5-HT3A and 5-HT3B), but the rat central nervous system seems to contain only one population of receptors formed by the 5-HT3A subunits only (Morales and Wang, 2002). Therefore, the data obtained using transfected X. laevis oocytes probably reflect the situation in the central nervous system, and it can be reasonably concluded that acetaminophen does not interact with 5-HT3 receptors in the dose range needed for its antinociceptive action. Under these conditions, the demonstrated blockade of the antinociceptive effect of acetaminophen by tropisetron might involve, at best, indirect effects at 5-HT3 receptors.
In vivo studies confirmed the complete prevention of the antinociceptive effect of acetaminophen by tropisetron but failed to reveal any clear-cut influence of the other two 5-HT3 receptor antagonists tested, granisetron and ondansetron, up to the doses of 10 µg/rat. At the highest dose used (20 µg/rat), granisetron significantly inhibited the action of acetaminophen 40 and 60 min after its administration. However, granisetron did not modify significantly the effect of acetaminophen on AUC scores. Nor did ondansetron. Furthermore, the specificity of these antagonists at such high doses can be questioned. Previous studies showed, for instance, that 1 µg of granisetron per rat was enough to strongly inhibit, in the same behavioral test, the antinociceptive effect of the potent 5-HT3 receptor agonist mCPBG (Bardin et al., 1997). In contrast, at this dose, neither granisetron nor ondansetron inhibited acetaminophen-induced antinociceptive effect, whereas tropisetron does at 0.5 and 1 µg/rat (Pélissier et al., 1996). From these data, which show differences between 5-HT3 receptor antagonists, as previously reported in clinical studies (e.g., Langlois et al., 1996), it can be concluded that 5-HT3 receptors are not involved in the antinociceptive effect of acetaminophen. It is interesting that Bardin et al. (1997) reached similar conclusions. At doses from 0.001 to 1 µg/rat, tropisetron inhibits the antinociceptive effect of 5-HT in the paw pressure test, whereas granisetron 0.1 and 1 µg/rat does not, supporting a non-5-HT3–mediated antinociceptive effect of 5-HT.
Tropisetron seems to have specific influences on the antinociceptive effect of acetaminophen and 5-HT. Moreover, it possesses a specific receptor profile; in addition to 5-HT3 receptor blockade, it binds to 5-HT4 receptors (Arranz et al., 1998), 
7 and 9 nicotinic receptors (Rothlin et al., 1999; Macor et al., 2001), and glycinergic receptors (Maksay et al., 1999). Therefore, the tropisetron-sensitive action of acetaminophen might involve other receptor types than the 5-HT3 receptors. The antisense study confirmed this hypothesis. The antinociceptive effect of the two highest selected doses of mCPBG, 1 and 10 µg/rat, was reduced by approximately 60% after AODN pretreatment, which induced a 30% reduction of 5-HT3A receptor labeling within the superficial laminae of the dorsal horn of the spinal cord. Such differences between the reduction of receptor density and that of its agonist action have already been described [for example, with opioid receptor (Pasternak and Standifer, 1995)]. The degree of down-regulation of receptors observed with AODNs depends on the sensitivity of the assay system used (for review, see Stone and Vulchanova, 2003). In the hypothesis where 5-HT3 receptors play a role in the antinociceptive action of acetaminophen, such AODN-induced down-regulation of spinal 5-HT3 receptors should have affected this drug action. However, after acetaminophen administration, animals pretreated with AODNs presented vocalization thresholds similar to those pretreated with saline or mismatch ODNs. These data further support the conclusion that spinal 5-HT3 receptors are not involved in the central mechanism underlying the antinociceptive action of acetaminophen.
However, the present results do not exclude the hypothesis of an indirect serotonergic mechanism of action of acetaminophen. This hypothesis is based on previous data evoking a mobilization by acetaminophen of endogenous serotonin (Tjolsen et al., 1991; Pini et al., 1996; Courade et al., 2001b) and showing that 5-HT receptor antagonists similarly influence the antinociceptive effect of 5-HT and that of acetaminophen (Courade et al., 2001c). Therefore, Chen and Bazan (2003) demonstrate an inhibition by acetaminophen of hippocampal synaptic plasticity via a 5-HT2 receptor and thus suggest the involvement of a release of endogenous 5-HT in this effect. However, the actual effect of acetaminophen on central 5-HT systems remains to be elucidated. In fact, complex interactions between 5-HT neurotransmission and other neurotransmitter systems might be evoked, which would explain the unique ability of tropisetron to prevent the antinociceptive action of acetaminophen. In this respect, particular attention should be focused on cholinergic and glycinergic neurotransmissions, because tropisetron acts at both nicotinic and glycinergic receptors, and these systems are clearly involved in pain modulation (Millan, 2002). Further work is thus needed to clarify the mechanism of the antinociceptive action of acetaminophen. In this context, the present results interestingly demonstrated that this mechanism involves a spinal site of action that can be uniquely affected by tropisetron through non–5-HT3 receptors.
|
Footnotes |
|---|
ABBREVIATIONS: AODN, antisense oligodeoxynucleotides; mCPBG, m-chlorophenylbiguanide; ODN, oligodeoxynucleotides; AUC, areas under the time course curves.
Address correspondence to: Alain Eschalier, INSERM/UdA E9904, Faculté de médecine, Place Henri-Dunant - B.P. 38, 63001 Clermont-Ferrand Cedex 1-France. E-mail: alain.eschalier{at}u-clermont1.fr
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Amin AR, Vyas P, Attur M, Leszczynska-Piziak J, Patel IR, Weissmann G, Abramson SB (1995) The mode of action of aspirin-like drugs: effects on inducible nitric oxide synthase. Proc Natl Acad Sci USA 92: 7926–7930.
Arranz B, Rosel P, San L, Sarro S, Navarro MA, and Marcusson J (1998) Characterization of the 5-HT4 binding site in human brain. J Neural Transm 105: 575–586.[CrossRef][Medline]
Bannwarth B, Netter P, Lapicque F, Gillet P, Pere P, Boccard E, Royer RJ, and Gaucher A (1992) Plasma and cerebrospinal fluid concentrations of paracetamol after a single intravenous dose of propacetamol. Br J Clin Pharmacol 34: 79–81.[Medline]
Bardin L, Jourdan D, Alloui A, Lavarenne J, and Eschalier A (1997) Differential influence of two serotonin 5-HT3 receptor antagonists on spinal serotonin-induced analgesia in rats. Brain Res 765: 267–272.[CrossRef][Medline]
Bonnefont J, Courade JP, Alloui A, and Eschalier A (2003) Mechanism of the antinociceptive effect of paracetamol. Drugs 63: 1–4.[Medline]
Bourinet E, Charnet P, Tomlinson WJ, Stea A, Snutch TP, and Nargeot J (1994) Voltage-dependent facilitation of a neuronal alpha 1C L-type calcium channel. EMBO (Eur Mol Biol Organ) J 13: 5032–5039.[Medline]
Chandrasekharan NV, Dai H, Roos KL, Evanson NK, Tomsik J, Elton TS, and Simmons DL (2002) COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/ antipyretic drugs: cloning, structure and expression. Proc Natl Acad Sci USA 99: 13926–13931.
Chen C and Bazan NG (2003) Acetaminophen modifies hippocampal synaptic plasticity via a presynaptic 5-HT2 receptor. Neuroreport 14: 743–747.[CrossRef][Medline]
Courade JP, Besse D, Delchambre C, Hanoun N, Hamon M, Eschalier A, Caussade F, and Cloarec A (2001a) Acetaminophen distribution in the rat central nervous system. Life Sci 69: 1455–1464.[CrossRef][Medline]
Courade JP, Caussade F, Martin K, Besse D, Delchambre C, Hanoun N, Hamon M, Eschalier A, and Cloarec A (2001b) Effects of acetaminophen on monoaminergic systems in the rat central nervous system. Naunyn Schmiedeberg's Arch Pharmacol 364: 534–537.[CrossRef][Medline]
Courade JP, Chassaing C, Bardin L, Alloui A, Eschalier (2001c) 5-HT receptor subtypes involved in the spinal antinociceptive effect of acetaminophen in rats. Eur J Pharmacol 432: 1–7.[CrossRef][Medline]
Dinchuk JE, Liu RQ, and Trzaskos JM (2003) COX-3: in the wrong frame in mind. Immunol Lett. 86: 121.[CrossRef][Medline]
Doucet E, Miquel MC, Nosjean A, Verge D, Hamon M, and Emerit MB (2000) Immunolabeling of the rat central nervous system with antibodies partially selective of the short form of the 5-HT3 receptor. Neuroscience 95: 881–892.[CrossRef][Medline]
Farivar RS, Chobanian AV, and Brecher P (1996) Salicylate or aspirin inhibits the induction of the inducible nitric oxide synthase in rat cardiac fibroblasts. Circ Res 78: 759–768.
Gelgor L, Cartmell S, and Mitchell D (1992) Intracerebroventricular micro-injections of non-steroidal anti-inflammatory drugs abolish reperfusion hyperalgesia in the rat's tail. Pain 50: 323–329.[CrossRef][Medline]
Hunskaar S, Fasmer OB, and Hole K (1985) Acetylsalicylic acid, paracetamol and morphine inhibit behavioral responses to intrathecally administered substance P or capsaicin. Life Sci 37: 1835–1841.[CrossRef][Medline]
Langlois A, Pascaud X, Junien JL, Dahl SG, and Riviere PJ (1996) Response heterogeneity of 5-HT3 receptor antagonists in a rat visceral hypersensitivity model. Eur J Pharmacol 318: 141–144.[CrossRef][Medline]
Laporte AM, Koscielniak T, Ponchant M, Verge D, Hamon M, and Gozlan H (1992) Quantitative autoradiographic mapping of 5-HT3 receptors in the rat CNS using [125I]iodo-zacopride and [3H]zacopride as radioligands. Synapse 10: 271–281.[CrossRef][Medline]
Macor JE, Gurley D, Lanthorn T, Loch J, Mack RA, Mullen G, Tran O, Wright N, and Gordon JC (2001) The 5-HT3 antagonist tropisetron (ICS 205–930) is a potent and selective alpha7 nicotinic receptor partial agonist. Bioorg Med Chem Lett 11: 319–321.[CrossRef][Medline]
Maksay G, Laube B, and Betz H (1999) Selective blocking effects of tropisetron and atropine on recombinant glycine receptors. J Neurochem 73: 802–806.[CrossRef][Medline]
Maricq AV, Peterson AS, Brake AJ, Myers RM, and Julius D (1991) Primary structure and functional expression of the 5-HT3 receptor, a serotonin-gated ion channel. Science (Wash DC) 254: 432–437.
Mestre C, Pélissier T, Fialip J, Wilcox G, and Eschalier A (1994) A method to perform direct transcutaneous intrathecal injection in rats. J Pharmacol Toxicol Methods 32: 197–200.[CrossRef][Medline]
Millan MJ (2002) Descending control of pain. Prog Neurobiol 66: 355–474.[CrossRef][Medline]
Mitchell JA, Akarasereenont P, Thiemermann C, Flower RJ, and Vane JR (1993) Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase. Proc Natl Acad Sci USA 90: 11693–11697.
Morales M and Wang SD (2002) Differential composition of 5-hydroxytryptamine3 receptors synthesized in the rat CNS and peripheral nervous system. J Neurosci 22: 6732–6741.
Pasternak GW and Standifer KM (1995) Mapping of opioid receptors using antisense oligodeoxynucleotides: correlating their molecular biology and pharmacology. Trends Pharmacol Sci 16: 344–350.[CrossRef][Medline]
Paul D, Yao D, Zhu P, Minor LD, and Garcia MM (2001) 5-Hydroxytryptamine3 (5-HT3) receptors mediate spinal 5-HT antinociception: an antisense approach. J Pharmacol Exp Ther 298: 674–678.
Pélissier T, Alloui A, Caussade F, Dubray C, Cloarec A, Lavarenne J, and Eschalier A (1996) Paracetamol exerts a spinal antinociceptive effect involving an indirect interaction with 5-hydroxytryptamine3 receptors: in vivo and in vitro evidence. J Pharmacol Exp Ther 278: 8–14.
Pini LA, Sandrini M, and Vitale G (1996) The antinociceptive action of paracetamol is associated with changes in the serotonergic system in the rat brain. Eur J Pharmacol 308: 31–40.[CrossRef][Medline]
Raffa RB and Codd EE (1996) Lack of binding of acetaminophen to 5-HT receptor or uptake sites (or eleven other binding/uptake assays). Life Sci 59: 37–40.
Randall LO and Selitto JJ (1957) A method for measurement of analgesic activity on inflamed tissue. Arch Int Pharmacodyn 61: 409–419.
Rothlin CV, Katz E, Verbitsky M, and Elgoyhen AB (1999) The 9 nicotinic acetylcholine receptor shares pharmacological properties with type A 
-aminobutyric acid, glycine and type 3 serotonin receptors. Mol Pharmacol 55: 248–254.
Ryu YS, Lee JH, Seok JH, Hong JH, Lee YS, Lim JH, Kim YM, and Hur GM (2000) Acetaminophen inhibits iNOS gene expression in RAW 264.7 macrophages: differential regulation of NF-kappaB by acetaminophen and salicylates. Biochem Biophys Res Commun 272: 758–764.[CrossRef][Medline]
Schwab JM, Schluesener HJ, and Laufer S (2003) COX-3: just another COX or the solitary elusive target of paracetamol? Lancet 361: 981–982.[CrossRef][Medline]
Stone LS and Vulchanova L (2003) The pain of antisense: in vivo application of antisense oligonucleotides for functional genomics in pain and analgesia. Adv Drug Deliv Rev 55: 1081–1112.[CrossRef][Medline]
Srikiatkhachorn A, Tarasub N, and Govitrapong P (1999) Acetaminophen-induced antinociception via central 5-HT2A receptors. Neurochem Int 34: 491–498.[CrossRef][Medline]
Tjolsen A, Lund A, and Hole K (1991) Antinociceptive effect of paracetamol in rats is partly dependent on spinal serotonergic systems. Eur J Pharmacol 193: 193–201.[CrossRef][Medline]
Warner TD, Giuliano F, Vojnovic I, Bukasa A, Mitchell JA, and Vane JR (1999) Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc Natl Acad Sci USA 96: 7563–7568.
Zeitz KP, Guy N, Malmberg AB, Dirajlal S, Martin WJ, Sun L, Bonhaus DW, Stucky CL, Julius D, Basbaum AI (2002) The 5-HT3 subtype of serotonin receptor contributes to nociceptive processing via a novel subset of myelinated and unmyelinated nociceptors. J Neurosci 22: 1010–1019.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
), mCPBG (
), acetaminophen (
), (5 to 32). The fitting of the mCPBG curve gives an EC50 of 7.8 µM. D, normalized currents evoked by 5-HT (10 µM) or mCPBG (30 µM) in the absence (
) of 0.3 mM acetaminophen.
