Remifentanil produces cross-desensitization and tolerance with morphine on the mu-opioid receptor

Neuropharmacology 73 (2013) 368e379

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Remifentanil produces cross-desensitization and tolerance with morphine on the mu-opioid receptor M. Nowoczyn a, N. Marie b, c, d, L. Coulbault a, M. Hervault a, A. Davis a, J.L. Hanouz a, S. Allouche a, * a

Université de Caen, Laboratoire de signalisation, Électrophysiologie et imagerie des lésions d’ischémie-reperfusion myocardique, UPRES EA 4650, IFR 146 ICORE, Avenue côte de Nacre, 14032 Caen, France Centre National de Recherche Scientifique, Unité Mixte de Recherche 8206, 4 Avenue de l’observatoire, 75006 Paris, France c Institut National de la Santé et de la Recherche médicale, U705, 4 Avenue de l’observatoire, 75006 Paris, France d Université Paris Descartes, Laboratoire de Neuropsychopharmacologie des Addictions, 4 Avenue de l’observatoire, 75006 Paris, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 September 2012 Received in revised form 26 May 2013 Accepted 7 June 2013

Remifentanil is a powerful mu-opioid (MOP) receptor agonist used in anaesthesia with a very short halflife. However, per-operative perfusion of remifentanil was shown to increase morphine consumption during post-operative period to relieve pain. In the present study, we aimed to describe the cellular mechanisms responsible for this apparent reduction of morphine efficacy. For this purpose, we first examined the pharmacological properties of both remifentanil and morphine at the MOP receptor, endogenously expressed in the human neuroblastoma SH-SY5Y cell line, to regulate adenylyl cyclase and the MAP kinase ERK1/2 pathway, their potency to promote MOP receptor phosphorylation, arrestin 3CFP (cyan fluorescent protein) recruitment and receptor trafficking during acute and sustained exposure. In the second part of this work, we studied the effects of a prior exposure of remifentanil on morphine-induced inhibition of cAMP accumulation, activation of ERK1/2 and analgesia. We showed that sustained exposure to remifentanil promoted a rapid desensitization of opioid receptors on both signalling pathways and a pretreatment with this agonist reduced signal transduction produced by a second challenge with morphine. While both opioid agonists promoted Ser375 phosphorylation on MOP receptor, remifentanil induced a rapid internalization of opioid receptors compared to morphine but without detectable arrestin 3-CFP translocation to the plasma membrane in our experimental conditions. Lastly, a cross-tolerance between remifentanil and morphine was observed in mice using the hot plate test. Our in vitro and in vivo data thus demonstrated that remifentanil produced a rapid desensitization and internalization of the MOP receptor that would reduce the anti-nociceptive effects of morphine. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Remifentanil Mu-opioid receptor Tolerance Desensitization Morphine Analgesia

1. Introduction

Abbreviations: MOP, mu-opioid receptor; DOP, delta-opioid receptor; GPCR, G protein-coupled receptor; MAP kinase, Mitogen-activated protein kinase; ERK1/2, Extracellular-regulated kinase 1/2; GFP, green fluorescent protein; CFP, cyan fluorescent protein; bFNA, b-funaltrexamine; NTI, naltrindole; DAMGO, [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin; MPE, maximal possible effect; GRK, G protein-coupled receptor kinase. * Corresponding author. Laboratoire de Biochimie, Centre Hospitalier et Universitaire, Avenue côte de nacre, 14033 Caen Cedex, France. Tel.: þ33 231065419; fax: þ33 231065172. E-mail addresses: [email protected] (M. Nowoczyn), nicolas.marie@ parisdescartes.fr (N. Marie), [email protected] (L. Coulbault), audrey.davis@ hotmail.fr (A. Davis), [email protected] (J.L. Hanouz), [email protected] (S. Allouche). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.06.010

Remifentanil, described as a mu-opioid (MOP) receptor selective agonist (James et al., 1991), is an ultra-short-acting opioid whose effects rapidly disappear once the perfusion is stopped due to a rapid metabolism by non-specific blood esterases (Glass et al., 1993). Consequently, remifentanil is widely used as analgesic in the per-operative period in association with propofol (see for review Scott and Perry, 2005). Additionally, remifentanil was reported to cause no delay of wakening and less respiratory depression than other opioids used in general anaesthesia (Dershwitz et al., 1995). Despite evident benefits, some studies pointed out acute tolerance following remifentanil infusion both in animals (Hayashida et al., 2003) and humans (Vinik and Kissin, 1998). For example, patients undergoing abdominal surgery under remifentanil perfusion showed significantly higher

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post-operative morphine consumption than patients who did not receive remifentanil (Guignard et al., 2000; Joly et al., 2005). Similar data were obtained in scoliosis surgery of adolescents by Crawford et al. (2006) who observed an increase in cumulative morphine consumption when using remifentanil during anaesthesia compared to morphine (Crawford et al., 2006). However, such observations were not confirmed although similar population and methods were used indicating that literature about remifentanilinduced tolerance is still controversial (Cortínez et al., 2001). It’s now well admitted that chronic opioid receptor stimulation induces desensitization characterized by a reduction of signal transduction. Opioid receptor desensitization would be a crucial step involved in tolerance as shown in arrestin 3 knockout mice. Indeed, the lack of this protein abolished MOP receptor desensitization and tolerance to the anti-nociceptive action of morphine (Bohn et al., 2000). In addition, arrestin 3 was recently shown to impair MOP receptor resensitization in locus ceruleus neurons contributing to morphine tolerance (Dang et al., 2011). Such a new role of arrestin 3 in MOP receptor resensitization and recycling was also confirmed by another group after chronic treatment of rat with morphine (Quillinan et al., 2011). Molecular mechanisms of opioid receptor desensitization have been extensively studied, and as for the majority of G protein-coupled receptors (GPCRs), they involve phosphorylation, recruitment of arrestins and receptor internalization (see for review Marie et al., 2006). Based on literature, we hypothesized that sustained remifentanil treatment would cause cross-desensitization of MOP receptor with morphine. In such conditions, post-operative pain would be relieved only when using high doses of morphine. To test our hypothesis, we examined MOP receptor signalling on the inhibition of adenylyl cyclase and the mitogen-activated protein (MAP) kinase pathways upon acute or sustained activation by remifentanil or morphine. Our experiments were conducted in the human neuroblastoma cell line SH-SY5Y which endogenously expresses MOP receptors. Opioid receptor trafficking was also examined by transfecting the SH-SY5Y cells with the MOP receptor fused to GFP2. Then, we studied the mechanisms of desensitization by focussing on MOP receptor phosphorylation (Ser375) and arrestin 3 translocation. Finally, cross-tolerance between remifentanil and morphine was evaluated in mice using the hot plate test. 2. Material and methods 2.1. Cell culture and transfection The human neuroblastoma cell lines SH-SY5Y and SK-N-BE were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma Aldrich, Saint Quentin Fallavier, France), supplemented with 10% (v/v) foetal calf serum (PAA laboratories, Les Mureaux, France), 1% (v/v) antibiotic/antimycotic mixture (PAA laboratories), 2 mM  L-glutamine (PAA laboratories) at 37 C, in a humidified atmosphere containing 5% CO2. Cells were harvested at 80e90% confluence in the following buffer (137 mM NaCl, 3 mM KCl, 6 mM Na2HPO4, 15 mM KH2PO4 and 0.5 mM EDTA, pH 7.5) and mechanically dissociated with a Pasteur pipette. For internalization and arrestin studies, SH-SY5Y cells were transfected with the Amaxa’s NucleofectorÔ technology (Kit V, program A-23) according to the manufacturer’s instructions (Lonza, Levallois-Perret, France) using the plasmid encoding for the MOP receptor fused to GFP2 (Choi et al., 2006) or for the arrestin 3 fused to Cyan Fluorescent Protein (CFP) (kindly given by Pr Stéphane Laporte, University of McGill, Canada), respectively. 2.2. Animals Male Swiss mice (Janvier, France) weighing 25 g the day of the experiment were housed 8 per cage, on a 12 h day/night cycle in a temperature-controlled room (21  2  C). Food and water were available ad libitum. Animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) as well as French law with the standard ethical guidelines. All animal care and experimental procedures were approved by the local ethics committee of the faculty of pharmacy (Université Paris Descartes, France).

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Every effort was made to minimize the number of animals used and their discomfort. 2.3. cAMP production measurements SH-SY5Y and SK-N-BE cells were seeded in 24-well plates at a density of 4.105 cells per well and loaded with 0.6 mCi [3H] adenine (Perkin Elmer, Courtaboeuf, France) overnight. cAMP accumulation was determined during 5 min at 37  C in the presence of 1 mM isobutylmethylxanthine, 40 mM forskolin and in the absence or in the presence of opioid ligands as described previously (Allouche et al., 1996). MOP- and DOP-receptors mediated adenylyl cyclase inhibition was assessed using morphine (Francopia, Paris, France), [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) (Sigma Aldrich) or remifentanil (UltivaÒ, GlaxoSmithKline, Marly-le-Roi, France) combined or not with opioid antagonists b-funaltrexamine (Tocris Bioscience, Lille, France) or naltrindole (Sigma Aldrich). In time-course experiments, cells were pretreated or not either with remifentanil or morphine during 5e120 min before measuring the cAMP accumulation. In crossdesensitization experiments, SH-SY5Y cells were exposed to a prior agonist then challenged with a second opioid drug without removing the medium. The [3H]cAMP content of each well was isolated by chromatography on acid alumina columns, mixed with 7 mL of scintillation mixture (Picofluor 40, Perkin Elmer) before assaying on a scintillation counter (Perkin Elmer). The percentage of inhibition was calculated according to the following formula : (1-(cpm agonist e cpm basal)/(cpm forskolin e cpm basal))  100, where cpm basal was determined in a medium containing only isobutylmethylxanthine and cpm forskolin in the presence of both isobutylmethylxanthine and forskolin. All experiments were carried out in triplicate. 2.4. Determination of the MAP kinase extracellular-regulated kinase (ERK)1/2 activation SH-SY5Y cells were cultured in 6-well plates until 90e100% confluence. After a 6e8 h period of serum starvation, cells were exposed or not (naive) to opioid ligands either during 5 min or from 5 to 120 min in time-course experiments at 37  C. In cross-desensitization experiments, cells were preincubated with a first opioid agonist then challenged with a second drug during 5 min at 37  C without removing the medium. ERK1/2 activation was determined by western blot as previously described using antibodies directed against phospho- and total ERK1/2 (Cell Signaling TechnologyÒ, Danvers, MA) (Aguila et al., 2007). Immunoreactive proteins were visualized by enhanced chemiluminescence (Western Lightning Chemiluminescence Reagent Plus, Perkin Elmer) using Fluor S Multimager. Densitometric analysis was performed using Quantity One software (Bio-Rad Laboratories, Marnes-la-Coquette, France). For each condition, we determined the ratio of phospho-ERK1/2/total ERK1/2 and results were normalized to the maximal effect except for cross-desensitization experiments in which 100% corresponded to the activation of ERK1/2 produced by the combination of both remifentanil and morphine. 2.5. Determination of Ser375 phosphorylation of the MOP receptor SH-SY5Y cells over-expressing MOP receptor-GFP2 were seeded in 6-well plates at a density of 106 cells per well and incubated overnight. Cells were then exposed or not (naive) to 10 mM remifentanil or morphine for 10 min at 37  C. Cell lysates were prepared as previously described (Aguila et al., 2007) and samples were resolved on 10% SDS-PAGE. After electroblotting, nitrocellulose sheets were incubated with either anti-P-Ser375 (1/1000 in TBS/Tween 0.1%/BSA 5%, Cell Signaling TechnologyÒ) or anti-GFP (1/5000 in TBS/Tween 0.1%/skimmed milk 5%, Cell Signaling TechnologyÒ). Immunoreactive proteins were quantified by densitometric analysis as described above for MAP kinase activation. For each condition, we determined the ratio P-Ser375 MOP receptor/MOP receptor-GFP2 and results were normalized to the maximal phosphorylation. 2.6. MOP receptor trafficking and arrestin 3 translocation experiments SH-SY5Y cells were transiently transfected with the MOP receptor-GFP2 or arrestin 3-CFP, then grown on alcohol-cleaned glass coverslips. Cells were exposed or not (naive) to opioid agonists, antagonist or a combination of both for different periods. For recycling experiments, the agonists were removed, cells were washed twice with DMEM and placed in agonist-free medium at 37  C. Then, cells were fixed using a fresh solution of 4% (w/v) paraformaldehyde for 15 min, rinsed with PBS and mounted. Images were obtained using a FluoView confocal laser microscope (Olympus, France), lens 60. 2.7. Behavioural experiments Analgesia was determined by the hot plate test after opioid agonist injection of 0.1 mL/10 g of bodyweight via the intraperitoneal (i.p.) route (Cordonnier et al., 2007). As previously described (Eddy and Leimbach, 1953), a glass cylinder (25 cm high, 20 cm diameter) was used to keep the mouse on the heated surface of the

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plate, which was kept at a temperature of 52  C (Panlab, Barcelona, Spain). The latency period until the mouse jumped was registered by means of a stop-watch (cutoff time 180 s). Percentage of analgesia was calculated using the following equation: % analgesia ¼ [(post-drug latency (s)  baseline latency (s))/(cut-off value (s)  baseline latency (s))]  100. 2.8. Statistical analysis All results are expressed as the mean  S.E.M. of n experiments. t-test, one-way ANOVA followed by the Bonferroni’s test or Dunnett’s test were used when appropriate to determine the statistical significance. EC50 or IC50 values and maximal effect of opioid ligands were determined by curve fitting of doseeresponse curves using GraphPad PrismÔ software.

3. Results 3.1. Remifentanil, morphine and DAMGO inhibit cAMP accumulation First, we evaluated the ability of three opioid agonists to inhibit adenylyl cyclase. Experiments were conducted in the human neuroblastoma SH-SY5Y cell line expressing both MOP and deltaopioid (DOP) receptors in a ratio of 4:1 (Noble et al., 2000). As shown in Fig. 1, remifentanil induced an inhibition of cAMP accumulation in a dose-dependent manner with a maximal of 46.2  3.8% at 1 mM and with an EC50 of 26.4  9.9 nM. Morphine also triggered a significant inhibition of cAMP but using concentrations higher than 100 nM. The inhibition reached a similar extent compared to remifentanil (39.5  4.1%) but with a weaker potency (EC50 of 554  33 nM) (P < 0.001, One-way ANOVA followed by Bonferroni’s test). DAMGO, known as a full agonist (Selley et al., 1998), promoted a significantly stronger inhibition of cAMP accumulation at 1 mM (69.6  8.4% for DAMGO vs 42.1  2.1% for remifentanil and 23.2  4.5% for morphine, P < 0.05, One-way ANOVA followed by Bonferroni’s test) but with a similar potency compared to remifentanil (EC50 of 24.5  5.2 nM) (Fig. 1). Even if remifentanil and morphine were described as MOP receptor selective agonists, we examined their effects on the DOP receptors as the SH-SY5Y cells contained both opioid receptors. For this purpose, we chose another human neuroblastoma, the SK-N-BE cell line which was demonstrated to express only DOP receptor (Allouche et al., 2000; Polastron et al., 1994). In this model, remifentanil was shown to promote only a modest inhibition

Fig. 1. Remifentanil, morphine and DAMGO inhibit cAMP accumulation in SH-SY5Y cells. SH-SY5Y cells were exposed during 5 min to increasing concentrations of either remifentanil, morphine or DAMGO (0.1 nMe10 mM) and cAMP accumulation was determined as described in material and methods. Data are the means  S.E.M of 3 independent experiments performed in triplicate. Doseeresponse curves were calculated with GraphPad PrismÔ software to determine both the maximal inhibition and EC50. **, P < 0.01, One-way ANOVA followed by Bonferroni’s test.

(20.7  4.1%) even at the highest concentration of 10 mM. In contrast, morphine displayed a greater efficacy with an inhibition of 55.8  3.0% obtained at 10 mM (P < 0.01, t-test) (Supplemental data 1). In order to demonstrate that remifentanil-induced cAMP inhibition was mediated specifically by the MOP receptors, SHSY5Y cells were exposed to 100 nM remifentanil (R-7) with increasing concentrations of the selective MOP receptor antagonist b-funaltrexamine (bFNA) (from 100 pM to 100 nM). As shown in Fig. 2A, the inhibition produced by remifentanil was gradually reversed when applying bFNA with a total blockade obtained at 100 nM bFNA and an IC50 of 6.65  3.30 nM. Similar experiments were conducted using 100 nM remifentanil combined with increasing concentrations of the selective DOP receptor antagonist naltrindole (NTI) from 1 nM to 1 mM. Only the highest concentration of 1 mM NTI was effective to antagonize the cAMP inhibition produced by 100 nM remifentanil (Fig. 2B). 3.2. Remifentanil and morphine induce MOP receptor desensitization on the cAMP pathway Second, we challenged SH-SY5Y cells either with 1 mM remifentanil or 10 mM morphine for various times (from 5 to 120 min) then the ability of MOP receptors to inhibit adenylyl cyclase was measured. As depicted on Fig. 3, the inhibition produced by remifentanil rapidly decreased with a significant reduction by more than 50% observed after 10 min pretreatment. After 30 min, a total desensitization of opioid receptors and even a superactivation of adenylyl cyclase were observed (Fig. 3). Same experiments were performed with morphine but with a different time-course since no desensitization was observed for short time exposure (5 and 10 min, data not shown). A significant reduction of the inhibitory action of this opioid agonist was obtained only after 60 min pretreatment and was total after 120 min (Fig. 3). 3.3. Remifentanil pretreatment produces a cross-desensitization of MOP receptors with morphine on the cAMP pathway Next, we examined whether a pretreatment of SH-SY5Y cells with remifentanil reduced the response to a second challenge with morphine and reciprocally. Concentrations of both opioid agonists close to the EC50 were chosen to avoid maximal response. Naive cells were treated either with a single agonist, 100 nM remifentanil (R-7) or 1 mM morphine (M-6), or both (R-7 þ M-6). Remifentanil or morphine promoted a similar inhibition on the adenylyl cyclase by 30%. When cells were exposed to a combination of both opioid agonists, no significant additive effect was evidenced (Fig. 4A and B). After 30 min pretreatment with remifentanil, no more inhibition of cAMP accumulation was measured (R-7 þ R-7 30 min, Fig. 4A). Such a reduction in the ability of remifentanil to inhibit adenylyl cyclase was not due to a degradation of the opioid agonist in the culture medium since the addition of freshly prepared agonist did not restore the inhibition (data not shown) demonstrating a total desensitization of MOP receptors. To avoid a strong superactivation of adenylyl cyclase (Avidor-Reiss et al., 1995), remifentanil (first challenge) was not removed from the medium and SH-SY5Y cells were exposed to morphine (second challenge). In such conditions, we observed that morphine was not able to induce inhibition of cAMP accumulation anymore (R-7 30 min þ M-6) suggesting that MOP receptors were cross-desensitized (Fig. 4A). The same experiments were conducted except that SH-SY5Y cells were first exposed to 1 mM morphine for 120 min to desensitize MOP receptors then to a second challenge with remifentanil. In such conditions, no more inhibition of cAMP accumulation was evidenced after the second challenge with 100 nM remifentanil (Fig. 4B).

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Fig. 2. bFNA but not naltrindole gradually reverses the inhibition of cAMP induced by remifentanil. SH-SY5Y cells were exposed during 5 mine100 nM remifentanil (R-7) with or without increasing concentrations of the selective MOP receptor antagonist bFNA (A, 100 pMe100 nM) or the selective DOP receptor antagonist NTI (B, 1 nMe1 mM) and cAMP accumulation was measured. Data are the means  S.E.M of 3 independent experiments performed in triplicate. Significance compared to remifentanil alone (R-7) is indicated: *, P < 0.05, **, P < 0.01, One-way ANOVA followed by Dunnett’s test.

3.4. Remifentanil pretreatment produces a cross-desensitization of MOP receptors with morphine on the MAP kinase ERK1/2 pathway As described for many GPCRs, opioid receptors promote phosphorylation of ERK1/2 upon their activation (Aguila et al., 2007; Belcheva et al., 1998). Since the response on this signalling pathway can be different to that produced on adenylyl cyclase as demonstrated for the adrenergic receptors (Galandrin and Bouvier, 2006), we studied the effects of remifentanil and morphine on ERK1/2 pathway. SH-SY5Y cells were challenged during 5 min with increasing concentrations of remifentanil and the level of activation, reflected by the ratio of phospho-ERK1/2/total ERK1/2, was measured. After normalization of data, we showed that remifentanil produced a dose-dependent increase in ERK1/2 activation with an EC50 of 212  65 nM (Fig. 5A). The level of activation produced at 10 mM was 4.5  0.3-fold over basal. In the presence of morphine, ERK1/2 activation was also dose-dependent with an EC50 of 1.71  0.39 mM. A 4.3  0.4-fold activation was observed at 10 mM morphine (Fig. 5B).

In time-course experiments, the maximal activation of ERK1/2 promoted by 10 mM remifentanil peaked at 5 min then decreased progressively (Fig. 6A). A significant reduction of ERK1/2 phosphorylation was evidenced from 15 min treatment. After 60 min exposure, MOP receptor desensitization was estimated at about 60% (Fig. 6A). When SH-SY5Y cells were challenged with 10 mM morphine, we also observed a maximal ERK1/2 activation at 5e 10 min followed by a significant decrease from 15 min to 60 min where a desensitization by 60% was measured (Fig. 6B). Then, we evaluated whether a first exposure to remifentanil reduced the response to a second challenge with morphine on the ERK1/2 phosphorylation. When SH-SY5Y cells were treated during 5 min either with 10 mM remifentanil (R-5), morphine (M-5) or a combination of both agonists (R-5 þ M-5), we observed almost the same extent of ERK1/2 activation (Fig. 7A and B). After a 60 minpretreatment with 10 mM remifentanil (R-5 60 min), a significant reduction by 50% of ERK1/2 phosphorylation was measured. When cells were first exposed during 60 min to 10 mM remifentanil then challenged with 10 mM morphine during 5 min (R-5 60 min þ M-5), a significant MOP receptor desensitization was observed compared to acute treatment (R-5 þ M-5) (Fig. 7A). Reciprocally, when cells were exposed during 60 min to 10 mM morphine (M-5 60 min), the MOP-receptor desensitization was about 70% (Fig. 7B). Next, SHSY5Y cells were first exposed to 10 mM morphine during 60 min then challenged during 5 min with 10 mM remifentanil (M-5 60 min þ R-5); in those conditions, remifentanil was not able to promote any ERK1/2 activation compared to the exposition of both agonists during 5 min (M-5 þ R-5) (Fig. 7B). 3.5. Remifentanil promotes phosphorylation and internalization of MOP receptors with no detectable arrestin 3 plasma membrane translocation

Fig. 3. Remifentanil and morphine induce MOP receptor desensitization on cAMP pathway. SH-SY5Y cells were pretreated or not (0 min) either with 1 mM remifentanil or 10 mM morphine for various times (from 5 min to 120 min). Then, the inhibitory effect of each opioid agonist was measured during 5 min as described in the material and methods section. Results were normalized to 100% corresponding to the inhibition obtained in naive cells. Data are the means  S.E.M of 3e5 independent experiments performed in triplicate. Significance compared to naive cells exposed to remifentanil and morphine is indicated respectively: **, P < 0.01, One-way ANOVA followed by Dunnett’s test and $$, P < 0.01, One-way ANOVA followed by Dunnett’s test.

Since we observed a differential MOP receptor desensitization on two signalling pathways upon remifentanil and morphine exposure, and it’s well admitted that phosphorylation and internalization of opioid receptors participate in desensitization (see for review Marie et al., 2006), we examined those two processes. First, we determined the phosphorylation level of MOP receptor at Ser375, which was described as the main amino acid to be phosphorylated by GRKs (Doll et al., 2011). SH-SY5Y over-expressing MOP receptor-GFP2 were exposed or not (naive) to 10 mM morphine or remifentanil during 10 min; such agonist concentrations produce a similar inhibition of cAMP accumulation (Fig. 1). As

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Fig. 4. Remifentanil induces cross-desensitization of MOP receptor with morphine on the cAMP pathway. SH-SY5Y cells were challenged either with 100 nM remifentanil (R-7) or 1 mM morphine (M-6) alone or a combination of both (R-7 þ M-6) during 5 min. A. In desensitization experiments, cells were exposed during 30 min either to remifentanil alone (R7 30 min) or remifentanil followed by morphine as a second challenge (R-7 30 min þ M-6). B. In desensitization experiments, cells were exposed during 120 min either to morphine alone (M-6 120 min) or morphine followed by remifentanil as a second challenge (M-6 120 min þ R-7). Data are the means  S.E.M of 5 independent experiments performed in triplicate. Significance compared to the inhibition produced by remifentanil alone (R-7), morphine alone (M-6) or the combination of both agonists (R-7 þ M-6) in naive cells is indicated: **, P < 0.01, One-way ANOVA followed by Bonferroni’s test.

depicted on Fig. 8, the two opioid agonists significantly increased phosphorylation of MOP receptors but surprisingly to a greater extent in the presence of morphine compared to remifentanil (Fig. 8A and B). Second, we studied trafficking of MOP receptors after remifentanil and morphine treatment. Since MOP receptor detection by immunocytochemistry in the neuroblastoma SH-SY5Y is difficult due to their low expression (see above), cells were transiently transfected with the human MOP receptor fused to the GFP2 and its

localization was determined by confocal microscopy. As depicted on Fig. 9, MOP receptor was distributed on the cell surface of naive SH-SY5Y cells. When cells were exposed to 100 nM remifentanil during various times (from 5 to 60 min, R 5 min to R 60 min), we observed a progressive reduction of the fluorescent signal at the plasma membrane with the concomitant formation of endocytic vesicles. The MOP receptor internalization was totally blocked by co-administration of 100 nM remifentanil and 10 mM naloxone during 60 min (R þ N 60 min) (Fig. 9), when naloxone (N) alone was

Fig. 5. Remifentanil and morphine induce a dose-dependent phosphorylation of ERK1/2. SH-SY5Y cells were serum-starved for 6e8 h then exposed to increasing concentrations of remifentanil or morphine (from 0.1 nM to 10 mM) during 5 min. Phospho- and total ERK1/2 were determined as described in material and methods. The MAP kinase activation level corresponds to the ratio pERK1/2/total ERK1/2. The maximal effect produced by remifentanil or morphine was chosen as 100%. Representative phospho- and total ERK1/2 westernblots are shown. Data are the means  S.E.M of 4e8 independent experiments.

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Fig. 6. Time-course of remifentanil and morphine-induced phosphorylation of ERK1/2. SH-SY5Y cells were serum-starved for 6e8 h then exposed or not (0 min) to 10 mM remifentanil (A) or morphine (B) for various times (5e60 min). Data are the means  S.E.M of 6 independent experiments. Significance compared to the maximal activation produced by agonist at 5 min is indicated: *, P < 0.05 or **, P < 0.01, One-way ANOVA followed by Dunnett’s multiple comparison tests. Representative phospho- and total ERK1/2 western-blots are shown.

devoid of any effect compared to naive cells (naive) (data not shown). In the presence of 1 mM morphine, we also observed a time-dependent MOP receptor internalization but to a lesser extent since after 120 min exposure (M 120 min), the fluorescence was localized both at the plasma membrane and in the cytosol (Fig. 9). As demonstrated for remifentanil, the morphine-induced MOP

receptor sequestration was totally blocked by co-administration with naloxone after 120 min (M þ N 120 min) (Fig. 9). In order to study the ability of the MOP receptor to recycle back to the plasma membrane, after 60 min for remifentanil or 120 min for morphine, the agonists were removed, cells were washed with DMEM and placed in agonist-free medium for various times. As shown in Fig. 9,

Fig. 7. Remifentanil pretreatment promotes cross-desensitization with morphine on the MAP kinase ERK1/2 pathway. SH-SY5Y cells were serum-starved for 6e8 h then exposed or not (none) to 10 mM remifentanil (R-5), morphine (M-5) or both (R-5 þ M-5) for 5 min. The maximal effect produced by the combination of both morphine and remifentanil was chosen as 100%. A. We determined ERK1/2 activation after 60 min pretreatment with 10 mM remifentanil followed or not (R-5 60 min, R-5) by a second challenge with 10 mM morphine during 5 min (R-5 60 min þ M-5). B. We determined ERK1/2 activation after 60 min pretreatment with 10 mM morphine followed or not (M-5 60 min, M-5) by a second challenge with 10 mM remifentanil during 5 min (M-5 60 min þ R-5). Data are the means  S.E.M of 3e5 independent experiments. Significance compared to the activation produced by a single agonist or remifentanil þ morphine are indicated respectively: *, P < 0.05, **, P < 0.01 and $$, P < 0.01, One-way ANOVA followed by Bonferroni’s test.

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Fig. 8. Remifentanil and morphine induce MOP receptor phosphorylation at Ser375. SH-SY5Y were transfected with a plasmid encoding for the human MOP receptor fused to GFP2. Forty eight hours post-transfection, cells were exposed or not () to 10 mM remifentanil (R) or morphine (M) during 10 min. A, Cells were lysed and immunoblotted with the anti-PSer375 antibody or anti-GFP to detect the phosphorylated and the total form of the MOP receptors, respectively. B, Immunoreactive bands were quantified by densitometry, the ratio P-Ser375 MOP receptor/total MOP receptor-GFP2 were determined and results were normalized to the maximal phosphorylation. Significance is indicated: **, P < 0.01 and or ***, P < 0.001 compared to the naives cells (), one-way ANOVA followed by Bonferroni’s test.

a partial receptor recycling was observed after 30 min for both agonists and slightly increased after 120 min (R 60 þ 120 min and M 120 þ 120 min). Third, the role of arrestin 3 in the regulation of MOP receptors was studied by confocal microscopy. SH-SY5Y cells were transfected with arrestin 3-CFP and its localization was observed at

different times (0, 5, 10, 15 and 30 min) after morphine (M), remifentanil (R) or DAMGO (D) treatment. In naive cells (N), arrestin 3-CFP displayed a diffuse cytoplasmic distribution pattern. Whatever the agonist used or the observation time, we were unable to detect any significant translocation of arrestin 3-CFP to the plasma membrane (Fig. 10A). We also transfected SH-SY5Y cells

Fig. 9. MOP receptor trafficking after remifentanil and morphine exposure. SH-SY5Y were transfected with a plasmid encoding for the human MOP receptor fused to GFP2. Forty eight hours post-transfection, cells were exposed or not (naive) to 100 nM remifentanil (R), 1 mM morphine (M), or a combination of agonist and 10 mM naloxone (N) (R þ N: remifentanil þ naloxone; M þ N : morphine þ naloxone) for various times (5e120 min). For recycling experiments, the agonist was removed and cells washed twice, then placed in agonist-free medium at 37  C for various times (30e120 min). After cell fixation, the cellular fluorescence localization was determined by confocal microscopy. Data are representative of 3 independent experiments.

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Fig. 10. Neither remifentanil, nor morphine, nor DAMGO promote arrestin 3-CFP translocation to the plasma membrane. A. The SH-SY5Y cells were transfected with a plasmid encoding for the arrestin 3 fused to CFP. Forty eight hours post-transfection, cells were exposed or not (naive, N) to 10 mM DAMGO (D), 10 mM morphine (M) or 1 mM remifentanil (R) for 10 min. B. The SH-SY5Y cells were co-transfected with plasmids encoding for the arrestin 3-CFP and the V2R-YFP. Forty eight hours post-transfection, cells were exposed or not (naive, N) to 1 mM arginine vasopressin (AVP) for 10 min. After cell fixation, the cellular fluorescence localization was determined by confocal microscopy. Data are representative of 2 independent experiments.

both with the arrestin 3-CFP and the vasopressin receptor 2 (V2R) fused to Yellow Fluorescent Protein (YFP), a GPCR known to form stable complex with arrestin 3 (Oakley et al., 2000). After a challenged with 1 mM arginine-vasopressin for 10 min, we could observe an obvious arrestin 3-CFP translocation and a colocalization of V2R and arrestin 3 at the plasma membrane (Fig. 10B, see merge). 3.6. Remifentanil promotes cross-tolerance with morphine in mice

we first checked that all the effects observed on the cAMP and the MAP kinase ERK1/2 pathways were only due to MOP receptor activation. bFNA, a selective MOP receptor antagonist, was able to dose-dependently reverse the inhibition of cAMP produced by remifentanil with a nanomolar concentration of IC50. This is in good agreement with the Ki of this antagonist reported by Raynor et al. (1994) and the data published by Liu et al. (2001) who showed that 100 nM bFNA totally blocked DAMGO-induced inhibition of cAMP accumulation. Furthermore, the partial blockade obtained

In order to confirm our in vitro data, in vivo experiments were conducted to evaluate the morphine-induced analgesia after remifentanil pretreatment using the hot plate test. In preliminary experiments, we showed that 5.5 mg/kg morphine administration induced a strong analgesia, and that 10 mg/kg remifentanil promoted 30% of maximal possible effect (MPE) in naive mice (data not shown). Remifentanil induced-anti-nociception was transient since 30 min after injection, no more analgesia could be measured (data not shown). Mice were challenged either with saline or 10 mg/kg remifentanil and after 30 min, we measured morphine-induced analgesia. As shown in Fig. 11, pretreatment with remifentanil significantly reduced the ability of morphine to promote analgesia compared to saline. 4. Discussion Remifentanil was described as a selective MOP receptor agonist (James et al., 1991; Scott and Perry, 2005) but since the SH-SY5Y cells express both MOP and DOP receptors (Noble et al., 2000),

Fig. 11. Remifentanil promotes cross-tolerance with morphine in vivo. Mice were pretreated with saline (Saline) or 10 mg/kg remifentanil (Remifentanil). After 30 min, animals received 5.5 mg/kg morphine and analgesia was measured with the hot plate test after 20 min. Significance is indicated: *, P < 0.05, Student’s t-test (n ¼ 8e10 animals/group).

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with only 1 mM NTI on the inhibition of adenylyl cyclase produced by remifentanil also indicates that DOP receptors are not involved. The concentration of NTI used is 50.000-folds the Ki for the DOP receptors and in such conditions, NTI is able to bind to MOP receptors (Raynor et al., 1994). To confirm such results, we used another human neuroblastoma cell line (SK-N-BE cells) but which contains only DOP receptors (Allouche et al., 2000; Polastron et al., 1994). We observed that micromolar concentrations of remifentanil promoted only a modest inhibition on the adenylyl cyclase. Since the SK-N-BE cells express more DOP receptors (100e200 fmol/mg of protein) than in the SH-SY5Y (20 fmol/mg of protein) and the MOP receptors are majority compared to DOP receptors in the SHSY5Y cell line, we can reasonably assume that only MOP receptors mediate the effects of remifentanil on both signalling pathways examined in the SH-SY5Y cells. When examining the two signalling pathways, we found that both opioid agonists were more potent to inhibit cAMP accumulation than to phosphorylate the MAP kinase ERK1/2. Yet, our data are in good agreement with a previous study performed in the SHSY5Y cells with morphine and we found that remifentanil and fentanyl display similar EC50 values for the inhibition of cAMP accumulation (Lambert et al., 1993). Furthermore, while DAMGO and remifentanil had a similar potency to inhibit adenylyl cyclase, we found that at 1 mM DAMGO was more efficacious than both remifentanil and morphine. As DAMGO was previously reported as a full MOP receptor agonist (Selley et al., 1998), our results suggest that remifentanil is, like morphine, a partial agonist on MOP receptor. When examining the MAP kinase ERK1/2 activation, we found that the magnitude of EC50 values for morphine and remifentanil were different from those calculated on the cAMP pathways. This suggests that either different mechanisms would be involved in the activation of the two signalling pathways or the two selective MOP receptor agonists would behave as biased agonists (Kenakin, 2011). In our conditions, the higher potency of remifentanil to phosphorylate ERK1/2 proteins than morphine would rather reflect the ability of remifentanil to preferentially stabilize the MOP receptor in a conformation enabling MAP kinase activation since both opioid agonists were reported to have similar affinity for the MOP receptor (Poisnel et al., 2006). We can also hypothesize that morphine-induced adenylyl cyclase inhibition would require more receptor occupancy than remifentanil. Recently, Rivero et al. (2012) demonstrated such differences between morphine vs DAMGO or etorphine both on [35S]GTPgS binding and on arrestin assay. Initially, we hypothesized that remifentanil would promote a strong and rapid desensitization of the MOP receptors hampering morphine to further activate the opioid receptor and trigger analgesia. Our data showed that remifentanil induced a faster and stronger desensitization than morphine on the two signalling pathways examined. This was more evident on the inhibition of adenylyl cyclase since a complete desensitization was observed after 30 min treatment with remifentanil compared to 120 min for morphine; this slow rate of desensitization produced by morphine has already been reported to occur in locus ceruleus neurons of rat on the MOP receptor-activated Kþ current (Bailey et al., 2009; Rivero et al., 2012). In contrast, Schulz et al. (2004) observed almost a similar time-course of MOP receptor desensitization in HEK cells when activated either by morphine or DAMGO. Such a differential opioid receptor desensitization was previously reported for MOP (see for review Marie et al., 2006) but also for DOP receptors (Aguila et al., 2007; Allouche et al., 1996, 1999; Lecoq et al., 2004) when comparing different agonists. It’s worth noting that after 30 min remifentanil and 120 min morphine pretreatment the cAMP level was greater compared to forskolin suggesting a superactivation of adenylyl cyclase. Such

phenomenon has been previously reported for MOP receptors expressed in heterogonous expression system (Avidor-Reiss et al., 1995) but also in SH-SY5Y cells (Levitt et al., 2011) and is generally observed when the agonist is rapidly removed after a longterm pretreatment or by the addition of an antagonist. We also previously reported adenylyl cyclase superactivation following chronic delta opioid receptor stimulation in the human neuroblastoma cell line SK-N-BE using agonists such as deltorphin I (Aguila et al., 2012). Several hypothesis have been proposed to explain such increase in adenylyl cyclase activity (see for review Watts, 2002): quantitative (decrease) or qualitative (changes in intracellular localization) modifications of Gai/o subunits, increase in Gbg dimers that would differentially regulate adenylyl cyclase isoforms and increase in Gas activity that would more effectively activate this enzyme. Although no superactivation is observed on the ERK1/2 pathway after long-term agonist exposure. This is probably due to different mechanisms of effectors activation (G proteins for adenylyl cyclase vs arrestins for MAP kinases), to the difference in terms of regulation of those effectors. Surprisingly, when regarding the MAP kinase ERK1/2 pathway, the difference between remifentanil- and morphine-induced desensitization was not observed anymore. This could be due to different mechanisms of regulation; on the cAMP pathway, kinases and arrestins were shown to participate in the MOP receptor desensitization (Johnson et al., 2006). In contrast, ERK1/2 activation by the MOP receptor was reported to implicate transactivation of the epidermal growth factor receptor (EGF-R) (Belcheva et al., 2001), and we can easily assume that this tyrosine kinase receptor would be the major regulatory site. This would explain that morphine and remifentanil displayed almost a similar time-course on the ERK1/2 phosphorylation. Indeed, MAP kinase activation produced both by those agonists occurred rapidly and after 60 min pretreatment, the desensitization level reached about 50%. A higher MOP receptor desensitization produced by morphine was previously reported in HEK transfected cells (Zheng et al., 2008) and in the SH-SY5Y cell line but using different experimental conditions (Bilecki et al., 2005). Both in in vivo and in vitro experiments, a pretreatment with remifentanil was shown to reduce the ability of morphine to further activate the two signalling pathways and to produce analgesia. Similar results were obtained when cells were first challenged with morphine then exposed to remifentanil. Moreover, the lack of additive effect observed when a combination of both agonists was used suggests that those ligands produced their effects at the same receptor. So, in such conditions, once the MOP receptor is rapidly desensitized by remifentanil the response to a further stimulation by morphine is reduced. From the classical model of GPCRs regulation (see for review (Marie et al., 2006)), opioid receptor desensitization involves their phosphorylation (Yu et al., 1997), uncoupling of receptors from their G proteins via arrestins (Bohn et al., 2000) and also their internalization (Walwyn et al., 2004). Several studies have demonstrated that MOP receptors are phosphorylated by multiple kinases (see for review Marie et al., 2006), and three amino acids located in the carboxy-terminus tail of MOP receptor were identified as the major phosphorylation site (Kouhen et al., 2001). Recently, Doll et al. (2011) suggested that Ser375 is phosphorylated in an agonist-dependant fashion while Ser363 is constitutively phosphorylated and phosphorylation of Thr370 is mediated by PKC (Doll et al., 2011). In our study, we showed that remifentanil and morphine both increased P-Ser375 as previously reported for morphine (Doll et al., 2011) but to a significantly greater extent in the presence of the alkaloid agonist compared to remifentanil. We can conclude that the different timecourse of MOP receptor desensitization on the cAMP pathway

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promoted by remifentanil and morphine is not directly linked to the phosphorylation of the Ser375. Previous works also reported a functional selectivity of opioid agonists at MOP receptor desensitization in vivo and showed the involvement of different kinases. The group of Chavkin demonstrated that GRK3 mediated MOP receptor desensitization upon sustained fentanyl exposure while the Jun N terminal kinase 2 (JNK2) is the main kinase involved in such regulatory mechanisms when morphine is used (Melief et al., 2010). Henderson’s laboratory showed both in HEK cells and in neurons that PKCa mediated morphine-induced MOP receptor desensitization while the GRK2 was responsible for the regulation of this opioid receptor when activated by DAMGO (Bailey et al., 2009; Johnson et al., 2006). In contrast, Met-enkephalin-induced MOP receptor desensitization in locus ceruleus neurons was shown to be independent of kinase activity (Arttamangkul et al., 2012). All those data indicate that mechanisms of opioid receptor desensitization are highly complex and largely depend on the agonist used. From our results and other studies (Doll et al., 2011), we can assume that remifentanil and morphine implicate at least a GRK that would phosphorylate the Ser375. Since phosphorylation of GPCRs by GRKs was described as the main signal to recruit arrestins at the plasma membrane which in turn promote uncoupling and internalization, we next examined the intracellular localization of arrestin 3 (also named b-arrestin 2). This arrestin was demonstrated to preferentially interact with the MOP receptor (Oakley et al., 2000) especially in the presence of morphine (Groer et al., 2011). However, we were unable to observe any translocation of arrestin 3-CFP upon either remifentanil, morphine or DAMGO exposure while the V2R, a GPCR known to form stable complex with arrestin 3 (Oakley et al., 2000), was able to promote an obvious arrestin 3-CFP translocation at the plasma membrane. These data are in good agreement with other studies reporting that in the presence of morphine MOP receptors poorly interact with arrestins (McPherson et al., 2010) and that MOP receptors activated by herkinorin are unable to promote arrestin 3GFP translocation to the plasma membrane (Groer et al., 2007). In our laboratory, we recently observed that while arrestin 2 was crucial for DOP receptor desensitization and internalization, no arrestin translocation was detectable (Aguila et al., 2012). Regarding remifentanil and to the best of our knowledge, no study has examined interaction between arrestins and the MOP receptors. However, fentanyl and alfentanil, two congeners of remifentanil, were shown to promote a strong arrestin 3 recruitment (McPherson et al., 2010). It’s also worthy to note that in studies demonstrating interactions between MOP receptors and arrestins or arrestin translocation, GRKs are over-expressed to increase interactions between receptor and arrestins (Groer et al., 2007; McPherson et al., 2010). Our data contrast with those published by Groer et al. (2011) where arrestins-GFP translocation was visualized in arrestins 2/3-KO mouse embryonic fibroblasts. Firstly, we can hypothesize that in our cellular model, only a small fraction of MOP receptor would be activated and would be able to recruit arrestins and that the endogenous arrestin 3 expressed in SH-SY5Y cells (Spartà et al., 2010) could compete with arrestin 3-CFP for the binding to MOP receptors. So, in such conditions, we couldn’t detect any plasma membrane translocation while in arrestins-depleted cells, this could be easily observed. Secondly, we can also speculate that arrestins are not involved in MOP receptor internalization as reported in knock-out arrestin 3 mice upon met-enkephalin exposure (Arttamangkul et al., 2012). Thirdly, arrestin experiments were conducted in SH-SY5Y cells transfected with arrestin 3CFP but endogenously expressing low levels of MOP receptors. Such experimental conditions wouldn’t be optimal to detect interactions between receptors and arrestins. So, even if we were unable to detect any plasma membrane translocation of arrestin 3-CFP we

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can’t completely rule out that remifentanil would promote arrestin interactions with MOP receptors. Using confocal microscopy, we observed a rapid loss of MOP receptors from the cell surface upon remifentanil exposure. So, it’s tempting to speculate that the reduction of active MOP receptors at the plasma membrane would play a major role in tolerance to morphine described in surgical patients and also in mice (present study). Only one study examined the remifentanil-induced MOP receptor internalization in rats (Trafton et al., 2000). In this paper, the systemic administration of remifentanil produced MOP receptor internalization visualized in the lamina II neurons of the spinal cord dorsal horn. This is in good agreement with our data showing strong and specific opioid receptor internalization upon remifentanil exposure. Fentanyl was also demonstrated to promote MOP receptor internalization when expressed in HEK293 cells (Zaki et al., 2000). However, in vivo administration of remifentanil during 30 min didn’t induce any significant change of MOP receptor expression measured either by RT-qPCR or western-blot in the dorsal root ganglia from 4 h to 7 days (Cabañero et al., 2009b). Such data are not in opposite with our study since we observed only a translocation of MOP receptors from cell surface into intracellular compartments; such redistribution of receptors is not necessary accompanied by quantitative modifications. Indeed, if remifentanil is removed, we were able to observe a partial recycling of MOP receptors. In contrast, morphine was described as an opioid agonist unable to trigger MOP receptor internalization (see for review Koch and Höllt, 2008). Our data clearly demonstrated that morphine could internalize the MOP receptor but with a slower time-course and to a lesser extent than remifentanil since after 120 min, we could still observe a partial labelling at the plasma membrane associated with intracellular vesicles. Another study demonstrated that morphine was also able to promote MOP receptor internalization in cultured striatum neurons (Haberstock-Debic et al., 2005). In the SH-SY5Y cells using binding experiments with [3H]CTAP, morphine was shown to decrease cell surface MOP receptors after 30 min indicating that this agonist is able to internalize opioid receptors in this cellular model (Horner and Zadina, 2004). 5. Conclusion In conclusion, few studies addressed the question about mechanisms responsible for the reduction of morphine analgesia after remifentanil perfusion (Cabañero et al., 2009a). We clearly demonstrated that remifentanil induces a cross-desensitization of MOP receptors with morphine on two different signalling pathways associated with a loss of membrane receptors. Such in vitro observations were also confirmed in vivo showing a cross-tolerance between remifentanil and morphine. To our knowledge, our data are the first to demonstrate that remifentanil produces molecular modifications at the MOP receptor level that would explain the higher morphine consumption when remifentanil is used in general anaesthesia. While our data suggest that MOP receptor internalization and desensitization would be a crucial step in such phenomenon, other mechanisms would occur such as DOP receptor down-regulation in dorsal root ganglia (Cabañero et al., 2009b) or the involvement of NMDA-receptor (Angst et al., 2003). Funding source Ministère de l’enseignement supérieur et de la recherche, Institut national de la santé et de la recherche médicale, Centre national de la recherche scientifique. There is no involvement of the financial support for the design of the study, for the collection, for analysis or interpretation of data, in the writing of the report or in the decision to submit the article for publication.

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Conflict of interest No conflict declared. Acknowledgements We would like to thank Dr Didier GOUX, Ph.D. (Centre de Microscopie appliquée à la biologie, Université de Caen, France) for his technical assistance concerning confocal microscopy. We thank Dr. Ping-Yee Law (Department of Pharmacology, University of Minnesota Medical School, Minneapolis, USA) for the pGFP2-N1hMOP plasmid and Pr Stéphane Laporte (University of McGill, Canada) for the plasmid containing the arrestin 3-CFP. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neuropharm.2013.06.010. References Aguila, B., Coulbault, L., Boulouard, M., Léveillé, F., Davis, A., Tóth, G., et al., 2007. In vitro and in vivo pharmacological profile of UFP-512, a novel selective deltaopioid receptor agonist; correlations between desensitization and tolerance. Br. J. Pharmacol. 152, 1312e1324. Aguila, B., Coulbault, L., Davis, A., Marie, N., Hasbi, A., Le Bras, F., et al., 2012. ßarrestin1-biased agonism at human d-opioid receptor by peptidic and alkaloid ligands. Cell. Signal. 24, 699e707. Allouche, S., Hasbi, A., Ferey, V., Sola, B., Jauzac, P., Polastron, J., 2000. Pharmacological delta1- and delta2-opioid receptor subtypes in the human neuroblastoma cell line SK-N-BE: no evidence for distinct molecular entities. Biochem. Pharmacol. 59, 915e925. Allouche, S., Polastron, J., Jauzac, P., 1996. The delta-opioid receptor regulates activity of ryanodine receptors in the human neuroblastoma cell line SK-N-BE. J. Neurochem. 67, 2461e2470. Allouche, S., Roussel, M., Marie, N., Jauzac, P., 1999. Differential desensitization of human delta-opioid receptors by peptide and alkaloid agonists. Eur. J. Pharmacol. 371, 235e240. Angst, M.S., Koppert, W., Pahl, I., Clark, D.J., Schmelz, M., 2003. Short-term infusion of the m-opioid agonist remifentanil in humans causes hyperalgesia during withdrawal. Pain 106, 49e57. Arttamangkul, S., Lau, E.K., Lu, H.-W., Williams, J.T., 2012. Desensitization and trafficking of m-opioid receptors in locus ceruleus neurons: modulation by kinases. Mol. Pharmacol. 81, 348e355. Avidor-Reiss, T., Bayewitch, M., Levy, R., Matus-Leibovitch, N., Nevo, I., Vogel, Z., 1995. Adenylylcyclase supersensitization in mu-opioid receptor-transfected Chinese hamster ovary cells following chronic opioid treatment. J. Biol. Chem. 270, 29732e29738. Bailey, C.P., Oldfield, S., Llorente, J., Caunt, C.J., Teschemacher, A.G., Roberts, L., et al., 2009. Involvement of PKC alpha and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of mu-opioid receptors in mature brain neurons. Br. J. Pharmacol. 158, 157e164. Belcheva, M.M., Szücs, M., Wang, D., Sadee, W., Coscia, C.J., 2001. mu-Opioid receptor-mediated ERK activation involves calmodulin-dependent epidermal growth factor receptor transactivation. J. Biol. Chem. 276, 33847e33853. Belcheva, M.M., Vogel, Z., Ignatova, E., Avidor-Reiss, T., Zippel, R., Levy, R., et al., 1998. Opioid modulation of extracellular signal-regulated protein kinase activity is rasdependent and involves Gbetagamma subunits. J. Neurochem. 70, 635e645.  ski, M.J., Bilecki, W., Zapart, G., Ligeza, A., Wawrzczak-Bargiela, A., Urban Przew1ocki, R., 2005. Regulation of the extracellular signal-regulated kinases following acute and chronic opioid treatment. Cell. Mol. Life Sci. 62, 2369e2375. Bohn, L.M., Gainetdinov, R.R., Lin, F.T., Lefkowitz, R.J., Caron, M.G., 2000. Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence. Nature 408, 720e723. Cabañero, D., Campillo, A., Célérier, E., Romero, A., Puig, M.M., 2009a. Pronociceptive effects of remifentanil in a mouse model of postsurgical pain: effect of a second surgery. Anesthesiology 111, 1334e1345. Cabañero, D., Célérier, E., García-Nogales, P., Mata, M., Roques, B.P., Maldonado, R., et al., 2009b. The pro-nociceptive effects of remifentanil or surgical injury in mice are associated with a decrease in delta-opioid receptor mRNA levels: prevention of the nociceptive response by on-site delivery of enkephalins. Pain 141, 88e96. Choi, H.S., Kim, C.S., Hwang, C.K., Song, K.Y., Wang, W., Qiu, Y., et al., 2006. The opioid ligand binding of human mu-opioid receptor is modulated by novel splice variants of the receptor. Biochem. Biophys. Res. Commun. 343, 1132e 1140. Cordonnier, L., Sanchez, M., Roques, B.P., Noble, F., 2007. Blockade of morphineinduced behavioral sensitization by a combination of amisulpride and RB101,

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