European Journal of Pharmacology 535 (2006) 169–171www.elsevier.com/locate/ejphar

Short communication

Role of gap junction in the expression of morphine-induced antinociception

Masami Suzuki 1, Minoru Narita ⁎, Atsushi Nakamura, Tsutomu Suzuki ⁎

Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan

Received 22 December 2005; accepted 20 January 2006Available online 7 March 2006

Abstract

The present study was undertaken to investigate whether gap junctional communication could be involved in morphine-inducedantinociceptive response using blockers of the gap junctional channel, carbenoxolone and Gap27. Intrathecal pretreatment with eithercarbenoxolone or Gap27 caused a dose-dependent attenuation of morphine-induced antinociception. Furthermore, the dose–response line formorphine-induced antinociception was shifted to the right by 2.53-fold following intrathecal treatment with carbenoxolone. These findingssuggest that gap-junctional-dependent communication in the mouse spinal cord may play, at least in part, a role in the expression of morphine-induced antinociception.© 2006 Elsevier B.V. All rights reserved.

Keywords: Morphine; Gap junction; Antinociception

1. Introduction

A gap junction is a unique channel that directly connects cellswith the interior of other cells. A gap junction enables theintercellular diffusion of molecules with a molecular mass of upto 1 kDa, including ions, cAMP, inositol (1,4,5)-triphosphate(IP3), ATP and small peptides (Giaume and Venance, 1998;Zonta et al., 2003). A gap junction is composed of a clusteredhemichannel, called a connexon, which forms aqueous conduitsthat link the intracellular compartments of coupled cells. Eachconnexon consists of a hexametric arrangement of gap junctionproteins called connexin (Kamermans et al., 2001; Bennett et al.,2003). Connexin forms at least 20 gene family members that areexpressed in many different cell types (Rash et al., 2001;Willecke et al., 2002). These channels can be gated in responseto various stimuli, including changes in voltage and intracellularpH (Saez et al., 2003). In the central nervous system, gap-junction-mediated intercellular communication between glialcells has long been thought to contribute to tissue homeostasis inthe brain. The homeostatic function includes the transport ofnutrients from the bloodstream to neurons, ‘spatial buffering’ of

⁎ Corresponding authors. Tel./fax: +81 3 5498 5628.E-mail addresses: narita@hoshi.ac.jp (M. Narita), suzuki@hoshi.ac.jp

(T. Suzuki).1 Tel./fax: +81 3 5498 5831.

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2006.01.038

K+ released into the extracellular space during neuronal exci-tation and uptake and dissipation of glutamate through gap-junction channels among astrocytes (De Pina-Benabou et al.,2001).

The stimulation of μ-opioid receptor by morphine canregulate a number of signaling pathways, including inhibi-tion of adenylate cyclase activity, activation of inwardlyrectifying K+ channels and blockade of Ca2+ entry throughvoltage-dependent Ca2+ channels (Childers, 1991). We pre-viously demonstrated that the endoplasmic IP3 receptor-mediated intracellular signaling pathway is implicated in theexpression of antinociceptive effect of morphine (Aoki et al.,2003). Therefore, the aim of the present study was toinvestigate whether the gap junctional channel in the spinalcord could be implicated in the expression of morphine-induced antinociception.

2. Materials and methods

The present study was conducted in accordance with theGuiding Principles for the Care and Use of Laboratory Animals,Hoshi University, as adopted by the Committee on AnimalResearch of Hoshi University, which is accredited by theMinistry of Education, Culture, Sports, Science and Technologyof Japan. Male ICR mice (Tokyo Laboratory Animals ScienceCo., Ltd.) were used in the present study.

Fig. 1. Effect of carbenoxolone on the antinociception produced bysubcutaneous (s.c.) injection of morphine. Intrathecal (i.t.) pretreatment withsaline and carbenoxolone was performed at 30 min before morphine injection.(A) Intrathecal pretreatment with carbenoxolone (0.03–1.00 nmol/mouse)attenuated the antinociception produced by morphine (10.0 mg/kg, s.c.) in aconcentration-dependent manner. ⁎⁎⁎Pb0.001: saline vs. 1.00 nmol carbenox-olone. (B) The dose–response curve for the antinociceptive effect of morphinein carbenoxolone-pretreated mice (closed circle, carbenoxolone; 1.00 nmol/mouse, ED50: 12.0 (8.18–17.5) mg/kg, s.c.) was significantly shifted to the rightcompared to saline-pretreated mice (open circle, ED50: 4.75 (2.91–7.73) mg/kg,s.c.). Antinociception was calculated as percentage of the maximum possibleeffect (%MPE) according to the following formula; %MPE=(test latency−pre-drug latency) / (cut-off time−pre-drug latency)×100. Each point represents themean±S.E.M. of 8–16 mice.

Fig. 2. Effect of Gap27 on the antinociception produced by s.c. injection ofmorphine. Intrathecal pretreatment with saline and Gap27 was performed at 30min before morphine injection. Intrathecal pretreatment with Gap27 (3.00 and10.0 nmol/mouse) attenuated the antinociceptive response produced by s.c.treatment with morphine in a concentration-dependent manner. ⁎⁎Pb0.01 and⁎⁎⁎Pb0.001 vs. saline-pretreated 5.00 mg/kg morphine group, ***Pb0.001 vs.saline-pretreated 10.0 mg/kg morphine group. Antinociception was calculated aspercentage of the maximum possible effect (% MPE) according to the followingformula; % MPE=(test latency−pre-drug latency) / (cut-off time−pre-druglatency)×100. Each point represents the mean 5±S.E.M. of 8–16 mice.

170 M. Suzuki et al. / European Journal of Pharmacology 535 (2006) 169–171

Morphine (Sankyo) and blockers of the gap junction, 3β-hydroxy-11-oxoolean-12-en-30-oic acid 3-hemisuccinate (car-benoxolone: Sigma-Aldrich) and Gap27 (amino-acid sequenceSRPTEKTIFII, Tocris), were dissolved in 0.9% sterile saline.

Intrathecal (i.t.) injection was performed as described byHylden and Wilcox (1980) using a 25 μl Hamilton syringe witha 30 gauge 1/2 in. needle. The injection volume was 4 μl for i.t.injection.

The morphine-induced antinociceptive response was evalu-ated by recording the latency to paw licking or tapping in thehot-plate test (55±0.5 °C). To prevent tissue damage, weestablished a 30 s cut-off time. The test was performed 30 min

after morphine treatment. Each animal served as its own control,and the latency to responses was measured both before and afterdrug administration. Antinociception was calculated as per-centage of the maximum possible effect (% MPE) according tothe following formula; % MPE= (test latency−pre-druglatency) / (cut-off time−pre-drug latency)×100. Antinocicep-tive response represents as the mean±S.E.M. of % MPE. TheED50 value was calculated by GraphPad Prism Programsversion 3.0 (GraphPad Software Inc.). The statistical signifi-cance of differences between groups was assessed with one-wayanalysis of variance (ANOVA) followed by the Bonferroni/Dunn multiple comparison test.

3. Results

To investigate the role of gap junctional communication inmorphine-induced antinociception, we examined the effects ofblockers of the gap junctional channel, carbenoxolone andGap27,on the μ-opioid receptor-mediated antinociceptive response usingthe hot-plate test. Intrathecal (i.t.) pretreatment with carbenox-olone (0.03–1.00 nmol/mouse) significantly attenuated the anti-nociceptive response produced by subcutaneous (s.c.) treatmentwithmorphine (10.0mg/kg) in a concentration-dependentmanner(⁎⁎⁎Pb0.001: saline-pretreated mice vs. 1.00 nmol carbenox-olone-pretreated mice, Fig. 1A). Furthermore, the dose–responsecurve for the antinociceptive effects ofmorphinewas significantlyshifted to the right by 2.53-fold following i.t. pretreatment withcarbenoxolone (1.00 nmol/mouse, Fig. 1B: ⁎⁎⁎ Pb0.001). Underthese conditions, carbenoxolone at the doses used in the presentstudy had no effects on the basal hot-plate latency after s.c. treat-ment with saline (data not shown). Like carbenoxolone,

171M. Suzuki et al. / European Journal of Pharmacology 535 (2006) 169–171

intrathecal pretreatment with Gap27 (3.00 and 10.0 nmol/mouse)significantly attenuated the antinociceptive response produced bys.c. treatment with morphine (5.00 and 10.0 mg/kg) in aconcentration-dependent manner (⁎⁎Pb0.01 and ⁎⁎⁎Pb0.001vs. saline-pretreated 5.00mg/kgmorphine group, ***Pb0.001 vs.saline-pretreated 10.0 mg/kg morphine group, Fig. 2).

4. Discussion

The central nervous system is composed of a closely associatednetwork of neurons and glial cells. For a long time, astrocyteswere considered to be limited to the structural, trophic andmetabolic support of neurons. Recent accumulating evidencesuggests that glial cells display rapid electrical responses toneuronal activity via gap junctional channels, which can triggerthe propagation of Ca2+ waves (Alvarez-Maubecin et al., 2000;Zonta et al., 2003). Ca2+ waves are mediated by the diffusion ofIP3 through gap junctions between cells and are also regulated bythe release of ATP though hemichannels (De Pina-Benabou et al.,2001; Galarreta and Hestrin, 2001).

Carbenoxolone is a moderately lipophilic glycyrrhetinic acidderivative that has been shown to act directly on gap junctions inbrain and other tissues to reduce conductance by up to 80%(Rozental et al., 2001). Furthermore, it has been reported thatGap27 peptides target the second extracellular loop of Cx40 andCx37/Cx43, which interrupt direct intracellular coupling in aconnexin-specific fashion without disrupting the structuralintegrity of gap junction plaques at points of intercellular contact(Chaytor et al., 1997, 1998). In the present study, we found that i.t.pretreatment with two different gap-junction blockers carbenox-olone and Gap27, which did not have any effects on thenociceptive threshold of the hot-plate response when injectedalone, caused a dose-dependent attenuation of morphine-inducedantinociception. Recent in vitro study using the purifiedastrocytes' primary culture shows that morphine failed to affectthe junctional permeability determined by dye transfer technique(Mantz et al., 1993). Consistent with these results, we previouslyreported thatmorphine had no direct effect on astrocytic activationin purified astrocytes, whereas it caused astrocytic activation inneuron/glia co-cultures (Narita et al., 2005, in press). Takentogether, these findings suggest that the gap junctional channel inthe spinal cord might be affected by morphine though thestimulation of μ-opioid receptor in neurons. Although gap-junction blockers do not clearly distinguish the two pathways,hemichannels and coupling between cells, these findings providenovel evidence that gap-junction channels in the spinal cord maybe, at least in part, implicated in the expression of morphine-induced antinociception.

Acknowledgments

This work was supported in part by grants from the Ministryof Health, Labour and Welfare, and the Ministry of Education,Culture, Sports, Science and Technology of Japan.

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