Journal of Pharmacological Sciences xxx (2016) 1e9 Contents lists available at ScienceDirect Journal of Pharmacological Sciences journal homepage: www.elsevier.com/locate/jphs Full paper Suppression of the acute upregulation of phosphorylated-extracellular regulated kinase in ventral tegmental area by a m-opioid receptor agonist is related to resistance to rewarding effects in a mouse model of bone cancer Atsushi Nakamura*, Hiroko Ono, Azusa Ando, Mikie Hinata, Kazuki Niidome, Shigeki Omachi, Gaku Sakaguchi, Shunji Shinohara Pain & Neuroscience, Drug Discovery & Disease Research Laboratory, Shionogi & Co., Ltd., 1-1, 3-chome, Futaba-cho, Toyonaka, Osaka 561-0825, Japan a r t i c l e i n f o a b s t r a c t Article history: Received 11 March 2016 Received in revised form September 2016 Accepted 13 November 2016 Available online xxx We investigated the mechanisms underlying the suppression of the rewarding effects of opioids using the femur bone cancer (FBC) mouse model The rewarding and antinociceptive effects of subcutaneously administered morphine and oxycodone in the FBC model mice were assessed using the conditioned place preference test and the von-Frey test In FBC mice, antinociceptive doses of morphine (30 mg/kg) and oxycodone (5 mg/kg) did not produce the rewarding effects but excessive doses of morphine (300 mg/kg) and oxycodone (100 mg/kg) did Western blot analyses revealed a transient and significant increase in phosphorylated-extracellular regulated kinase (p-ERK) levels in ventral tegmental area (VTA) after the administration of morphine in sham-group Interestingly, in FBC group, a regular dose of morphine did not increase p-ERK levels but a high dose of morphine caused an increase in p-ERK level after administration The rewarding effects of a regular dose of and a high dose of morphine in the sham-operation and FBC model, respectively, were significantly inhibited by the MEK inhibitor The suppression of p-ERK might result in resistance to these rewarding effects under the conditions of bone cancer © 2016 The Authors Production and hosting by Elsevier B.V on behalf of Japanese Pharmacological Society This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/) Keywords: Antinociception Bone cancer pain Extracellular regulated kinase Opioid Rewarding effect Introduction Although opioids have a constellation of side effects related to their potential for abuse and/or addiction in opioid naïve condition, a growing body of clinical evidence has shown that when opioids are appropriately used to control pain in cancer patients, psychological dependence is not a major concern (1) The femur bone cancer (FBC) model is useful as a cancer-related pain paradigm, because the model-induced pain and the pathological changes involve bone destruction and nerve compression that are observed within a few weeks after the tumor implantation Abbreviations: GABA, g-aminobutyric acid; CPP, conditioned place preference; FBC, femur bone cancer; NAC, nucleus accumbens; p-ERK, phosphorylated-extracellular regulated kinase; VTA, ventral tegmental area * Corresponding author E-mail address: atsushi_nakamura@shionogi.co.jp (A Nakamura) Peer review under responsibility of Japanese Pharmacological Society (2) Our previous studies have demonstrated the antinociceptive efficacy of the opioids using the FBC pain model (2,3) However, little is known regarding psychological dependence to opioids under cancer-related pain conditions including the FBC model The few published studies that employed non-cancer pain models, have been reported that morphine fails to induce psychological dependence in rodents (4e6) These experimental data support the clinical experience that if morphine is used appropriately under pain conditions then morphine-induced psychological dependence is unlikely to be a problem The mesolimbic dopaminergic system that projects from the ventral tegmental area (VTA) to the nucleus accumbens (NAC) is thought to be a critical brain region involved in the development of psychological dependence to opioids (7) Opioids activate the mopioid receptors that are located on g-aminobutyric acid (GABA) ergic neurons in the VTA, which results in the inhibition of these GABAergic and a subsequent increase in dopaminergic activity in the NAC (8) Therefore, the VTA is considered to be a crucial brain http://dx.doi.org/10.1016/j.jphs.2016.11.004 1347-8613/© 2016 The Authors Production and hosting by Elsevier B.V on behalf of Japanese Pharmacological Society This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article in press as: Nakamura A, et al., Suppression of the acute upregulation of phosphorylated-extracellular regulated kinase in ventral tegmental area by a m-opioid receptor agonist is related to resistance to rewarding effects in a mouse model of bone cancer, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.11.004 A Nakamura et al / Journal of Pharmacological Sciences xxx (2016) 1e9 region when attempting to understand the m-opioid receptor function in psychological dependence to opioids In the present study, we found that the optimal antinociceptive doses of morphine and oxycodone did not produce rewarding effects under the FBC model, and that one of the likely mechanisms underlying this suppression is the inhibition of the acute upregulation of phosphorylated extracellular regulated kinase (p-ERK) levels by opioids The present study utilized C3H/HeN mice weighing 18 ± 23 g (CLEA Japan, Inc., Tokyo, Japan) The animals were housed in a room maintained at 23 ± C under a 12 h light/dark cycle with ad libitum access to water and food Each animal was used only once All procedures for the animal experiments were approved by the Animal Care and Use Committee of Shionogi Research Laboratories (Osaka, Japan) in agreement with the internal guidelines for animal experiments and in adherence to the ethical policies of Shionogi & Co., Ltd ethical policy, and were performed according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines membranes, and incubated in blocking buffer (5% skim milk in Trisbuffered saline, 0.1% Tween 20) for 60 The membranes were incubated with an anti-p-ERK antibody (Phospho-p44/42 MAPK [Erk1/2] [Thr202/Tyr204] [D13.14.4E] XP® Rabbit mAb, 1:2000 dilution in blocking buffer; Cell Signaling technology) at C for 16 h, and then incubated with peroxidaselabeled anti-rabbit IgG (111-036-003, 1:5000 dilution in blocking buffer; Jackson Immuno Research Laboratories, West Grove, PA, USA) at room temperature for h The membrane-bound antibodies were detected using the ECL Plus Western Blotting Detection Reagent (GE Healthcare Life Sciences, Tokyo, Japan) and the Luminescent Image Analyzer (LAS-3000; Fujifilm, Tokyo, Japan) Antibodies of anti-p-ERK were then stripped using the stripping buffer (Restore TM Plus Western blot stripping buffer, Thermo) by incubating the membranes for 30 The absence of signal was checked by incubating the membrane with the peroxidase-labeled anti-rabbit IgG and ECL Plus Western Blotting Detection Reagent Membranes were then incubated in a blocking buffer for 60 and incubated with anti-ERK antibody ([p44/42 MAPK] [Erk1/2] [137F5] Rabbit mAb, 1:1000 dilution in blocking buffer; Cell Signaling technology) at C for 16 h The ERK signals were detected in the same manner as p-ERK and the signal intensities of each band were assessed using Multi Gauge software (Fujifilm, Tokyo, Japan) 2.2 The FBC mouse model 2.6 Immunohistochemistry The FBC mouse model of pain was prepared and implemented as previously described (2) Briefly, NCTC2472 tumor cells (American Type Culture Collection; Manassas, VA, USA) were injected into the medullary cavity of the left distal femur of the C3H/HeN mice Opioid-induced changes in the p-ERK immunoreactivity were assessed with immunohistochemistry Five minutes after administration of morphine, mice were immediately anaesthetized with isoflurane, perfused intracardially with PBS and fixed with 4% paraformaldehyde Midbrain including VTA was isolated, post-fixed and placed overnight in 30% sucrose The specimen frozen in OCT compound (Sakura Finetechnical, Tokyo, Japan) was cut into 10 mm sections using a cryostat For p-ERK1/2 detection, sections were heated using a microwave oven at 600 W in 10 mM citrate buffer (pH 6) for and incubated with rabbit anti-pERK1/2 antibody (1:500; Cell Signaling, Danvers, MA, USA) at C overnight After washing by PBS, the sections were reacted with Alexa Fluor 594conjugated anti-rabbit IgG antibody (1:500; Invitrogen) at room temperature for h, mounted with Vectashield (Vector Laboratory, Burlingame, CA, USA) and coverslipped Materials and methods 2.1 Animals 2.3 CPP test Place conditioning studies were conducted using a shuttle box (15 30 15 cm, w l h; KN-80; Natsume Seisakusyo Co., Ltd., Tokyo, Japan) as previously described (5) The place conditioning paradigm consisted of three phases: the pre-conditioning test, drug conditioning and the post-conditioning test 2.4 von-Frey test Allodynia-like behavior was evaluated by paw withdrawal response to tactile stimuli of a series of von-Frey monofilaments (pressure: 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6 and g) The upedown method of von-Frey monofilament test (2) was used in the present study 2.5 Western blot analyses Opioid-induced changes in the p-ERK protein levels were assessed with Western blot analyses The mouse VTA was quickly removed after decapitation and homogenized in lysis buffer (640 mM sucrose and 0.1 M HEPES) containing a complete miniEDTA-free tablet (Roche, Indianapolis, IN, USA) with a highvelocity revolution homogenizer The homogenates were centrifuged at 400 g at C for 10 min, the supernatants were centrifuged at 50,000 g at C for 30 min, and then the membrane fractions were collected The protein concentrations in the samples were determined using a BCA Protein Assay-Reducing Agent Compatible kit (Thermo Fisher Scientific K.K., Yokohama, Japan) according to the manufacturer's protocol The protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis (SDSPAGE), transferred onto polyvinylidene difluoride (PVDF) 2.7 [Tylosil-3,5-(3)H(N)]-[D-Ala(2),N-Me-Phe(4),Gly-ol(5)] enkephalin ([3H]DAMGO) and Guanosine-50 -o-(3-thio) triphosphate ([35S]GTPgS) binding assay [3H]DAMGO saturation binding assay and Guanosine-50 -o-(3thio) triphosphate [35S]GTPgS binding assay were used to measure the maximum number of binding sites (Bmax) and bind to half the receptor sites at equilibrium (Kd) for m-opioid receptor and Gprotein activation as previously described (3) 2.8 Drugs Morphine (morphine hydrochloride) and oxycodone (oxycodone hydrochloride) were obtained from Shionogi & Co., Ltd; they were dissolved in 0.9% physiological saline (Otsuka Pharmaceutical Co Inc., Tokyo, Japan) for the in vivo experiments and dissolved in assay buffer for the in vitro experiments The MEK inhibitor U0126 was purchased from SigmaeAldrich Co LLC., and dissolved in 10% dimethyl sulfoxide (DMSO; SigmaeAldrich) for the in vivo experiments The volume used for the s.c administrations was 0.1 mL per 10 g body weight Please cite this article in press as: Nakamura A, et al., Suppression of the acute upregulation of phosphorylated-extracellular regulated kinase in ventral tegmental area by a m-opioid receptor agonist is related to resistance to rewarding effects in a mouse model of bone cancer, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.11.004 A Nakamura et al / Journal of Pharmacological Sciences xxx (2016) 1e9 2.9 Statistical analysis The data were shown as mean ± standard error of mean (SEM) SAS software (ver 8) and GraphPad Prism 4.0 program were used to perform the statistical analyses The statistical significance of differences among groups were assessed by a two-way ANOVA followed by Dunnett's or Bonferroni multiple comparison test A probability value (p) of