Suppression of the acute upregulation of phosphorylated extracellular regulated kinase in ventral tegmental area by a opioid receptor agonist is related to resistance to rewarding effects in a mouse model of bone ca

Full paperSuppression of the acute upregulation of phosphorylated-extracellular agonist is related to resistance to rewarding effects in a mouse model of bone cancer Atsushi Nakamura*, H

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Full paper

Suppression of the acute upregulation of phosphorylated-extracellular

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

Article history:

Received 11 March 2016

Received in revised form

9 September 2016

Accepted 13 November 2016

Available online xxx

Keywords:

Antinociception

Bone cancer pain

Extracellular regulated kinase

Opioid

Rewarding effect

a b s t r a c t

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)

5 min 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

5 min 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

licenses/by-nc-nd/4.0/)

1 Introduction

Although opioids have a constellation of side effects related to

their potential for abuse and/or addiction in opioid nạve condition,

a growing body of clinical evidence has shown that when opioids

are appropriately used to control pain in cancer patients,

psycho-logical 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

patho-logical changes involve bone destruction and nerve compression

that are observed within a few weeks after the tumor implantation

(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 psy-chological dependence in rodents(4e6) These experimental data support the clinical experience that if morphine is used appropri-ately 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 m -opioid 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

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.

Contents lists available atScienceDirect Journal of Pharmacological Sciences

j o u r n a l h o m e p a g e :w w w e l s e v i e r c o m / l o c a t e / j p h s

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/ ).

Journal of Pharmacological Sciences xxx (2016) 1e9

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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

ef-fects under the FBC model, and that one of the likely mechanisms

underlying this suppression is the inhibition of the acute

upregu-lation of phosphorylated extracellular regulated kinase (p-ERK)

levels by opioids

2 Materials and methods

2.1 Animals

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 ± 1 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

2.2 The FBC mouse model

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

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 1 g) The up

e-down 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

mini-EDTA-free tablet (Roche, Indianapolis, IN, USA) with a

high-velocity revolution homogenizer The homogenates were

centri-fuged at 400g at 4C for 10 min, the supernatants were

centri-fuged at 50,000g at 4C 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

(SDS-PAGE), transferred onto polyvinylidene diﬂuoride (PVDF)

membranes, and incubated in blocking buffer (5% skim milk in Tris-buffered saline, 0.1% Tween 20) for 60 min

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 4C for 16 h, and then incubated with peroxidase-labeled anti-rabbit IgG (111-036-003, 1:5000 dilution in blocking buffer; Jackson Immuno Research Laboratories, West Grove, PA, USA) at room temperature for 1 h The membrane-bound anti-bodies were detected using the ECL Plus Western Blotting Detection Reagent (GE Healthcare Life Sciences, Tokyo, Japan) and the Lumi-nescent Image Analyzer (LAS-3000; Fujiﬁlm, Tokyo, Japan) Anti-bodies of anti-p-ERK were then stripped using the stripping buffer (Restore TM Plus Western blot stripping buffer, Thermo) by incu-bating the membranes for 30 min 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 min and incubated with anti-ERK antibody ([p44/42 MAPK] [Erk1/2] [137F5] Rabbit mAb, 1:1000 dilution in blocking buffer; Cell Signaling technology) at 4 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)

2.6 Immunohistochemistry

Opioid-induced changes in the p-ERK immunoreactivity were assessed with immunohistochemistry Five minutes after admin-istration 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 10mm 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 5 min and incubated with rabbit anti-pERK1/2 antibody (1:500; Cell Signaling, Danvers, MA, USA) at 4C overnight After washing by PBS, the sections were reacted with Alexa Fluor 594-conjugated anti-rabbit IgG antibody (1:500; Invitrogen) at room temperature for 1 h, mounted with Vectashield (Vector Laboratory, Burlingame, CA, USA) and coverslipped

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-(3-thio) triphosphate [35S]GTPgS binding assay were used to mea-sure the maximum number of binding sites (Bmax) and bind to half the receptor sites at equilibrium (Kd) form-opioid receptor and G-protein activation as previously described(3)

2.8 Drugs

Morphine (morphine hydrochloride) and oxycodone (oxyco-done 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 experi-ments The volume used for the s.c administrations was 0.1 mL per

10 g body weight

A Nakamura et al / Journal of Pharmacological Sciences xxx (2016) 1e9 2

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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

dif-ferences among groups were assessed by a two-way ANOVA

fol-lowed by Dunnett's or Bonferroni multiple comparison test A

probability value (p) of <0.05 was considered to be statistically

signiﬁcant

3 Results

3.1 Suppression of the rewarding effects of antinociceptive doses of

opioids in the FBC mouse model

The FBC model mice exhibited a marked decrease in the paw

withdrawal threshold on the ipsilateral side 14 days after

tumor-implantation, and did not show any tumor metastasis into brain

(2) Thus, the rewarding and antinociceptive effects of

subcutane-ously administration of morphine and oxycodone in mice were

examined using CPP and von-Frey tests under the condition of the

FBC model of pain

In the sham-operated mice, morphine (1e30 mg/kg, s.c.)

pro-duced a dose-dependent increase in the preference for the

drug-associated place time in the CPP test (Fig 1A) However, while

the administration of morphine (10 and 30 mg/kg, s.c.) resulted in

a marked and signiﬁcant place preference compared to saline

conditioning in the sham-operated mice, this morphine-induced

(30 mg/kg, s.c.) place preference was not observed in

tumor-implanted mice (Fig 1A) Based on these ﬁndings, a

dose-dependent experiment investigating the effects of morphine

with a minimum dose of 30 mg/kg was conducted in

tumor-implanted mice Only a very high dose of morphine (300 mg/kg,

s.c.) produced a similar place preference in tumor-implanted mice

that 30 mg/kg morphine did in sham-treated mice (Fig 1A)

Similarﬁndings were observed when using oxycodone

Oxyco-done (0.3e100 mg/kg, s.c.) produced a dose-dependent place

preference in both the sham-operated and tumor-implanted mice,

but the dose-dependent curve shifted dramatically to the right in

the FBC model mice compared to the sham-operated mice

(Fig 1B) Taken together, these results indicate that the rewarding

potencies of both morphine and oxycodone are reduced in the FBC model

Next, the antinociceptive effects of morphine and oxycodone were assessed in FBC model mice using the von-Frey test In the tumor-implanted mice, both morphine (3e30 mg/kg, s.c.) and oxycodone (1e10 mg/kg, s.c.) reversed the decrease in paw with-drawal thresholds in a dose-dependent manner, and the effects were restored to the level of the sham group at a dose of 30 mg/kg for morphine and a dose of 5 mg/kg for oxycodone, respectively (Fig 2A and B), which is essentially consistent with previous ﬁnd-ings(2) Taken together, these results suggest that antinociceptive doses of these two opioids did not produce the rewarding effects in the FBC model Theseﬁndings may be correlated with the clinical phenomenon in which the appropriate use of opioids for cancer patients does not result in psychological dependence

3.2 Suppression of the acute upregulation of p-ERK in the VTA by opioids under the FBC mouse model

To investigate the mechanisms underlying the suppression of the rewarding effects of opioids in the FBC mouse model, we investigated changes in p-ERK levels in the VTA It has been sug-gested that p-ERK is a major factor associated with the develop-ment of opioid dependence(9) However, changes in the pattern of p-ERK expression following the administration of opioids under physiological conditions have yet to be well-studied

The changes in p-ERK protein levels were evaluated by Western blot analyses during the time course study following the adminis-tration of morphine (10 and 300 mg/kg, s.c.) in the FBC model mice Compared to the administration of saline, a single administration of morphine (10 mg/kg, s.c.) produced a signiﬁcant increase in p-ERK protein levels in the VTA of sham-operated mice at 5 min (Fig 3A) However, at 30 min after a single administration of morphine (10 mg/kg, s.c.), this upregulation of p-ERK in the VTA was no longer observed in the sham-operated mice (Fig 3B)

In the VTA of tumor-implanted mice, neither a single adminis-tration of morphine (10 mg/kg, s.c.) nor saline inﬂuenced p-ERK protein levels at either 5 (Fig 3A) or 30 min (Fig 3B) Only a high dose of morphine (300 mg/kg, s.c.) produced a signiﬁcant increase

in p-ERK protein levels after a single administration at 30 min (Fig 3B) The upregulation of p-ERK in tumor-implanted mice at

Fig 1 Conditioned place preference for morphine and oxycodone under the FBC mouse model FBC mice were tested 13 days after tumor implantation Ordinate: mean difference in times spent in the compartment between the post-conditioning test and the pre-conditioning test Immediately after the subcutaneous administration of (A) morphine (1e300 mg/kg) or (B) oxycodone (0.3e100 mg/kg), the mice were placed and conditioned in either compartment for 1 h The ﬁlled square and open triangle indicate the sham-operated and tumor-implanted groups, respectively Each curve represents the mean ± SEM for 13e16 mice *p < 0.05 vs the saline-treated tumor-implanted group or ## p < 0.01,

# p < 0.05 vs the saline-treated sham group (two-way ANOVA, Dunnett's multiple comparison test).

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Fig 2 Antinociceptive effects of morphine and oxycodone under the FBC mouse model FBC mice were tested 14 days after tumor implantation Either (A) morphine (3e30 mg/ kg) or (B) oxycodone (1e10 mg/kg) was subcutaneously administered at 30 min or 15 min prior to the measurement of the paw withdrawal threshold in the von Frey monoﬁlament test The ﬁlled and open columns indicate the sham-operated and tumor-implanted groups, respectively Data represent the mean ± SEM of 6e8 mice ***p < 0.001 vs the saline-treated tumor-implanted group or ### p < 0.001, # p < 0.05 vs the saline-treated sham group (two-way ANOVA, Dunnett's multiple comparison test).

Fig 3 p-ERK levels in the VTA induced by morphine under the FBC mouse model Western blot analyses showed the changes in p-ERK protein levels following the subcutaneous administration of morphine (10 or 300 mg/kg) in the VTA Morphine was administered in a single (A, B) or twice of three days (C, D) injection and the membrane fractions of the lower midbrain, including the VTA region, were prepared either 5 min (A, C) or 30 (B, D) min after administration The brain tissue samples were analyzed with a SDS-PAGE procedure and the gel images illustrate a representative result; similar results were obtained in 4e8 independent experiments The black, gray, and white columns represent the relative protein levels in the groups treated with saline and morphine, respectively (the mean protein level in the saline-treated sham group was deﬁned as 1.0) The mean ± SEM of 4e8 independent experiments are shown ***p < 0.001 vs the saline-treated sham group or # p < 0.05 vs the saline-treated tumor-implanted group (two-way ANOVA, student's t-test).

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5 min after the single administration of 300 mg/kg morphine was

similar to the upregulation of p-ERK in sham-operated mice after

the single administration of 10 mg/kg morphine

We also included a chronic opioid administration protocol that

employed a schedule of two administrations every 3 days based on

the timing of the CPP paradigm We conﬁrmed the change in p-ERK

levels following the chronic administration of morphine in the VTA

using Western blot analyses There were no signiﬁcant differences

observed among the groups at either 5 (Fig 3C) or 30 min (Fig 3D)

after the last injection In the immunohistochemistry, the p-ERK

positive cells in VTA were relatively low in saline treated

sham-operated and tumor-implanted group (Fig 4) A single

adminis-tration of morphine (10 mg/kg, s.c.) showed an increase in p-ERK

positive cells in VTA of sham-operated mice at 5 min, and this

in-crease by morphine was not observed in tumor-implanted group

(Fig 4) These results indicate that the transient increase in p-ERK

levels in the VTA at 5 min after a single administration of morphine

is an integral aspect of understanding the inﬂuence of p-ERK in the

development of psychological dependence Furthermore, the acute

upregulation of p-ERK by optimal doses of morphine was

completely suppressed by tumor implantation

3.3 Effects of an MEK inhibitor on the rewarding and antinociceptive effects of opioids in the FBC mouse model

We also investigated the role of MEK signaling (upstream of ERK signaling) on the rewarding effects of opioids using the MEK in-hibitor U0126 The rewarding effects of opioids are modulated by the i.c.v administration of U0126 (4), and thus we assessed whether the U0126 (0.1e10 nmol/mouse, i.c.v.) would inhibit the morphine-induced place preference Morphine (0.1e10 nmol/ mouse, i.c.v.) resulted in a dose-dependent increase in place pref-erence (Fig 5A), that was dose-dependently and signiﬁcantly attenuated by U0126 (0.1e10 nmol/mouse, i.c.v.) (Fig 5B) Furthermore, up-regulation of p-ERK level by morphine (10 nmol/ mouse, i.c.v.) was signiﬁcantly abolished by U0126 (10 nmol/mouse, i.c.v.; Fig 5C) These results suggest that the morphine-induced place preference observed was positively linked with the MEK/ ERK pathway

Next, the inﬂuence of U0126 on the rewarding effects of opioids was investigated in the FBC model In the sham-operated mice, the place preference induced by morphine (10 mg/kg, s.c.) was signif-icantly inhibited by pretreatment with U0126 (10 nmol/mouse,

Fig 4 p-ERK immunoreactivity in the VTA induced by morphine under the FBC mouse model Typical images of immunohistochemistry showed the p-ERK-expressing neurons following the subcutaneous administration of saline or morphine (10 mg/kg) in the VTA of sham-operated and tumor-implanted mouse Green signals indicate p-ERK; the arrow shows positive cells Scale bar represents 50mm.

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i.c.v.) Although a high dose of morphine (300 mg/kg, s.c.) produced

place preference in tumor-implanted mice, this preference was also

signiﬁcantly attenuated by U0126 (Fig 6A) Similar to morphine,

the place preferences induced by oxycodone (3 mg/kg for sham

group, and 100 mg/kg for tumor-implanted group) were also

inhibited by U0126 (10 nmol/mouse, i.c.v.;Fig 6B) Theseﬁndings

suggest that MEK-related ERK signaling inﬂuences opioid reward,

and that the upregulation of p-ERK levels in VTA may trigger the

development of opioid-related rewarding effects in the FBC mouse model

The inﬂuence of U0126 on the antinociceptive effects of opioids was also assessed in the FBC model A 10 nmol/mouse adminis-tration of U0126 was selected for this assay because this particular dose clearly inhibited the rewarding effects of opioids in the pre-vious experiments of the present study Under these conditions, both the antinociceptive effects of morphine (30 mg/kg, s.c.) and

Fig 5 Effects of MEK-ERK signaling on the conditioned place preference for morphine (A) Dose response for morphine and (B) the role of the MEK inhibitor U0126 on the place preference for morphine assessed using the conditioned place preference paradigm Ordinate: mean difference in times spent in the compartment between the post-conditioning test and the pre-post-conditioning test The mice were treated with U0126 (0.1e10 nmol/mouse, intracerebroventricular [i.c.v.]) 10 min prior to morphine administra-tion Immediately after the i.c.v administration of morphine (0.1e10 nmol/mouse), the mice were placed and conditioned in either compartment for 1 h The ﬁlled and open columns indicate the sham-operated and tumor-implanted groups, respectively Each column represents the mean ± SEM of 13e16 mice **p < 0.01, *p < 0.05 vs the saline group, # p

< 0.05 vs the vehicle-morphine group (two-way ANOVA, Dunnett's multiple comparison test) (C) The role of the MEK inhibitor U0126 on p-ERK by morphine in the VTA assessed using the Western blot analyses Western blot analyses showing the changes in p-ERK protein levels The mice were treated with U0126 (10 nmol/mouse, i.c.v.) 10 min prior to morphine (10 nmol/mouse, i.c.v.) administration Immediately after the i.c.v administration of morphine (0.1e10 nmol/mouse), and mouse VTA samples were prepared 10 min after administration The brain tissue samples were analyzed with a SDS-PAGE procedure and the gel images illustrate a representative result; similar results were obtained in 8 in-dependent experiments The black, gray, and white columns represent the relative protein levels in the groups (the mean protein level in the vehicle-saline group was deﬁned as 1.0) The mean ± SEM of 4 independent experiments are shown **p < 0.01 vs the vehicle-saline group (two-way ANOVA, student's t-test).

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oxycodone (5 mg/kg, s.c.) did not signiﬁcantly differ from these

effects in mice pre-treated with U0126 (Fig 7) These results

sug-gest that, unlike the rewarding effects, MEK-related ERK signaling

did not play a major role in the antinociceptive effects of opioids in

the present study

3.4 Effects of G-protein signaling in the VTA on the rewarding

effects of opioids in the FBC mouse model

We hypothesized that the agonist activity of opioids atm-opioid

receptor would be dramatically reduced in the VTA of the FBC

model mice Thus, we investigated the total number ofm-opioid

receptors in the sham-operated and tumor-implanted mice by

measuring the Bmax and Kd of these receptors via [3H]DAMGO

saturation binding to cell membranes prepared from the VTA

(Fig 8A) The Bmax and Kd values were calculated using a Scatchard plot analysis, which revealed that the Bmax of [3H]DAMGO signif-icantly decreased by 36% in tumor-implanted mice relative to sham-operated mice but that the Kd of [3H]DAMGO binding did not signiﬁcantly differ between the sham-operated and tumor-implanted mice (Fig 8A) These ﬁndings indicate that the total amount ofm-opioid receptors in the tissue membranes from the VTA was reduced in the FBC model

Under these conditions, changes in the abilities of morphine and oxycodone to activate G-proteins were assessed in the lower midbrain area, including the VTA, in sham-operated and tumor-implanted mice by monitoring the binding of [35S]GTPgS to mem-branes The administration of either morphine (108e10e5M) or oxycodone (108e10e5M) resulted in a concentration-dependent

increase in the binding of [35S]GTPgS to lower midbrain

Fig 6 Role of MEK-ERK signaling in the opioid-induced place preference under the FBC mouse model FBC mice were tested 13 days after tumor implantation Ordinate: mean difference in times spent in the compartment between the post-conditioning test and the pre-conditioning test The mice were treated with U0126 (10 nmol/mouse, intra-cerebroventricular [i.c.v.]) 10 min prior to the subcutaneous administration of (A) morphine (10 or 300 mg/kg) or (B) oxycodone (3 or 100 mg/kg) Immediately after the administration of morphine or oxycodone, the mice were placed and conditioned in either compartment for 1 h The ﬁlled and open columns indicate the vehicle and U0126 groups, respectively Each column represents the mean ± SEM of 13e16 mice *p < 0.05, **p < 0.01 vs the morphine- or oxycodone-treated sham groups or # p < 0.05, ### p < 0.001 vs the morphine- or oxycodone-treated tumor-implantation groups (two-way ANOVA, Dunnett's multiple comparison test).

Fig 7 Role of MEK-ERK signaling in opioid-induced antinociception under the FBC mouse model FBC mice were tested 14 days after tumor implantation and morphine or oxycodone was administered 30 min or 15 min prior to the measurement of the paw withdrawal threshold in the von Frey monoﬁlament test The mice were treated with U0126 (10 nmol/mouse, intracerebroventricular [i.c.v.]) 10 min prior to the subcutaneous administration of morphine (30 mg/kg) or oxycodone (5 mg/kg) The ﬁlled and open columns indicate the vehicle and U0126 groups, respectively Data represent the mean ± SEM of 6 mice **p < 0.01, *p < 0.05 vs the saline-treated tumor-implanted group or ### p < 0.001 vs the saline-treated sham group (two-way ANOVA, Dunnett's multiple comparison test).

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membrane preparations from sham-operated mice (Fig 8B and C).

On the other hand, the levels of [35S]GTPgS binding in this area that

were stimulated by morphine and oxycodone in the

tumor-implanted mice were signiﬁcantly lower than the levels observed

in sham-operated mice (Fig 8B and C) Theseﬁndings indicate that

the opioid-inducedm-opioid receptor-mediated functional

activa-tion in the VTA was dramatically attenuated in the FBC mouse

model of pain

4 Discussion

The primary ﬁndings of the present study were that

anti-nociceptive doses of opioids did not produce rewarding effects in

the FBC mouse model of pain and that this effect is likely supported

by the inhibition of the acute upregulation of p-ERK levels in the

VTA by opioids

Morphine produces relatively low rewarding effects under

chronic pain states such as inﬂammatory and neuropathic pain

(4e6) Interestingly, the mechanisms underlying the suppression

of the rewarding effects of opioids differ among pain types For

example, under an inﬂammatory pain state, the upregulation of

kappa (k)-opioid receptors and/or dynorphin (an endogenousk

-opioid ligand) function due to chronic inﬂammatory pain or

chronic m-opioid receptor agonist therapy can decrease

psychological dependence tom-opioid receptor agonists(6) Un-der the neuropathic pain state, the selective and sustained acti-vation of mesolimbic b-endorphin (an endogenous m-opioid ligand) may be an important proximal mechanism supporting the suppression of the rewarding effects ofm-opioid receptor agonists (10) In the case of FBC pain state, the total amount ofm-opioid receptors on tissue membranes was reduced in the brain regions and spinal cord(3) Based on this evidence, a state of bone cancer pain may lead to reductions inm-opioid receptor function in the VTA that could, at least in part, result from the sustained release

of mesolimbicb-endorphin due to the neuropathic component of bone cancer

We also found that the inhibition of the ERK pathway was positively associated with the avoidance of opioid reward whereas the acute antinociceptive effects of these drugs were unaffected by

an MEK inhibitor in the FBC model Theseﬁndings indicate that the role of ERK may differ in terms of the rewarding and acute anti-nociceptive effects of opioid drugs Although it has been observed that a similar regulation of p-ERK levels is induced by opioids in pain-related brain regions under the FBC pain model (e.g., peri-aqueductal gray [PAG], thalamus, and spinal cord), MEK inhibitors

do not inﬂuence the acute antinociceptive effects of the opioids (11) Thus, it appears that the opioid-induced attenuation in the upregulation of p-ERK in the FBC mouse model results in the

VTA

0 20 40 60 80

Tumor-implanted

Bmax (fmol/mg protein)

Kd (nM)

Tumor-implanted 56.21 *** 1.348

-20 0 20 40 60 80

100

Sham Tumor-implanted

Concentration of oxycodone (logM)

+++

###

-20 0 20 40 60 80

100

Sham Tumor-implanted

***

Concentration of morphine (logM)

Fig 8 Number ofm-opioid receptors andm-opioid receptor function on VTA cell membranes under the FBC mouse model (A) Saturation curves for the specific binding of [ 3 H] DAMGO on VTA cell membranes prepared from sham-operated and FBC model mice Tissue samples were collected 14 days after the sham operation (Sham) or tumor implantation (tumor-implanted) and the membranes prepared from the VTA were used for the binding assay [ 3 H]DAMGO binding was examined using concentrations of 0.5e16 nM and specific binding was defined as the difference in binding observed in the absence and presence of 10mM of unlabeled [ 3 H]DAMGO Each value represents the mean ± SEM of three in-dependent experiments (8 mice/sample in each experiment) Kd and Bmax of [ 3 H]DAMGO in the VTA The values were determined from the saturation curve and the Scatchard plot analysis; at least 6 concentrations were used for each analysis ***p < 0.001 vs the sham group (two-way ANOVA, Dunnett's multiple comparison test) Concentration-response curve for (B) morphine (108e10 e5 M) and (C) oxycodone (108e10 e5 M) on the binding of [ 35 S]GTPgS to membranes obtained from the lower midbrain regions, including the VTA, of sham-operated and tumor-implanted mice The membranes were prepared at 14 days after tumor implantation The filled square and open triangle indicate the sham-operated and tumor-implanted groups, respectively Each symbol represents the mean ± SEM of three independent experiments (4 mice/sample in each experiment).

F (1,28) ¼ 21.84; ***p < 0.001 vs the sham morphine group or F (1,28) ¼ 14.20; ### p < 0.001 vs the sham oxycodone group (two-way ANOVA) þþþ p < 0.001 or þ p < 0.05 vs., the sham group (Bonferroni multiple comparison post-test).

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speciﬁc suppression of opioid reward without affecting

antinociception

We detected morphine-induced changes in p-ERK levels using

Western blot analyses and immunohistochemical study but the

speciﬁc cells in the VTA that were possibly targeted by opioids

remain undetermined Although the observed changes may have

been mediated by the opioid-induced activation of m-opioid

re-ceptors, p-ERK levels are known to be inﬂuenced by a variety of

molecules For example, the direct and indirect actions ofm-opioid

receptor agonists on the cell membrane must be considered(12) In

the case of the same action on the cell membrane, the activation of

m-opioid receptor results in an upregulation of p-ERK via the Gq

protein/PKC pathway(13), GRKs/b-arrestin2 pathway(14)and the

receptor tyrosine kinase transactivation pathway(15) Based on such

reports, it is possible that many molecules upstream of p-ERK are

involved in the suppression of opioid reward in the FBC mouse

model A time-course study may contribute towards a more

com-plete understanding of the activities of reward-related upstream

molecules of p-ERK that are inﬂuenced by opioids because the

ki-netic patterns of ERK activation may differ among these pathways

For instance, in the angiotensin 2 and parathyroid receptors, the fast

activation of p-ERK is mediated via G-proteins whereas the slow

activation of p-ERK is mediated byb-arrestins(16) Furthermore,b

-arrestin-2 does not positively inﬂuence opioid-induced rewarding

effects(17) Although the actions ofm-opioid receptor-independent

p-ERK pathways cannot be ruled out as factors involved in the

present results, the inhibition of the acute upregulation of p-ERK by

opioids may be mediated, at least in part, by them-opioid

receptor-related Gq/PKC pathway in the FBC mouse model

In conclusion, the present study raises the possibility that bone

cancer-related pain in mice may cause an inhibition of

opioid-induced p-ERK activation in the VTA, which results in a

suppres-sion of the rewarding effects of these drugs When opioids are used

appropriately their rewarding effects in terms of psychological

dependence are not a major issue These results provide important

evidence suggesting that opioids can be used clinically to control

pain in the absence of psychological dependence

Conﬂict of interest

All of the authors are employees of Shionogi& Co., Ltd, the

manufacture of oxycodone and morphine

Acknowledgements

No funding is associated with this manuscript

References

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Tiêu đề	Suppression of the acute upregulation of phosphorylated extracellular regulated kinase in ventral tegmental area by a opioid receptor agonist is related to resistance to rewarding effects in a mouse model of bone cancer
Tác giả	Atsushi Nakamura, Hiroko Ono, Azusa Ando, Mikie Hinata, Kazuki Niidome, Shigeki Omachi, Gaku Sakaguchi, Shunji Shinohara
Trường học	Shionogi & Co., Ltd.
Chuyên ngành	Pharmacological Sciences
Thể loại	Research article
Năm xuất bản	2016
Thành phố	Osaka

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Số trang	9
Dung lượng	1,52 MB