logy 536 (2006) 85–92www.elsevier.com/locate/ejphar

European Journal of Pharmaco

Effects of dextrorotatory morphinans on α3β4 nicotinic acetylcholinereceptors expressed in Xenopus oocytes

Jun-Ho Lee a,1, Eun-Joo Shin b,1, Sang Min Jeong a, Jong-Hoon Kim a, Byung-Hwan Lee a,In-Soo Yoon a, Joon-Hee Lee a, Sun-Hye Choi a, Sang-Mok Lee a, Phil Ho Lee c,

Hyoung-Chun Kim b,⁎, Seung-Yeol Nah a,⁎

a Department of Physiology, College of Veterinary Medicine, Konkuk University, Institute of Biomedical Science and Technology, and Bio/Molecular InformaticsCenter, 143-701 Konkuk University, Seoul, Republic of Korea

b Neurotoxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Koreac Department of Chemistry, Kangwon National University, Chunchon 200-701, Republic of Korea

Received 8 September 2005; received in revised form 23 January 2006; accepted 21 February 2006Available online 2 March 2006

Abstract

We previously demonstrated that dextromethorphan (DM; 3-methoxy-17-methylmorphinan) analogs have neuroprotective effects, and a recentreport showed that DM reduces the adverse effects of morphine and blocks α3β4 nicotinic acetylcholine receptors, a major target of anti-addictiveagents. Here, we investigated the effects of DM, three of its analogs (DF, 3-methyl-17-methylmorphinan; AM, 3-allyloxy-17-methoxymorphian;and CM, 3-cyclopropyl-17-methoxymorphinan) and one of its metabolites (HM; 3-methoxymorphinan), on neuronal α3β4 nicotinic acetylcholinereceptor channel activity expressed in Xenopus laevis oocytes, using the two-microelectrode voltage clamp technique. We found that intraoocyteinjection of neuronal α3 and β4 nicotinic acetylcholine receptor subunit cRNAs elicited an inward current (IACh) in the presence of acetylcholine.Co-treatment with DM, DF, AM, CM or HM inhibited IACh in a dose-dependent, voltage-independent and reversible manner. The IC50 values forDM, DF, AM, CM and HM were 19.5±5.2, 15.8±4.5, 16.3±1.7, 10.1±2.8, and 13.5±4.0 μM, respectively. The order of potency for theinhibition of IACh was CM N HM N DF = AM N DM in oocytes expressing α3β4 nicotinic acetylcholine receptors. The inhibitions of (IACh) byDM, DF and HM, AM and CM were non-competitive. These results indicate that AM, CM and HM could be novel non-competitive agentsregulating α3β4 nicotinic acetylcholine receptor channel activity.© 2006 Elsevier B.V. All rights reserved.

Keywords: Dextromethorphan; Dextromethorphan analog; α3β4 nicotinic acetylcholine receptor; Xenopus oocyte

1. Introduction

Dextromethorphan (DM; 3-methoxy-17-methylmorphinan),a non-narcotic morphinan derivative, is closely related in

⁎ Corresponding authors. Kim is to be contacted at Neurotoxicology Program,College of Pharmacy, Kangwon National University, Chunchon 200-701,Republic of Korea. Tel.: +82 33 250 6917; fax: +82 33 255 7865. Nah,Department of Physiology, College of Veterinary Medicine, Konkuk University,Institute of Biomedical Science and Technology, and Bio/Molecular InformaticsCenter, 143-701 Konkuk University, Seoul, Republic of Korea. Tel.: +82 2 4504154; fax: +82 2 450 3039.

E-mail addresses: kimhc@kangwon.ac.kr (H.-C. Kim),synah@konkuk.ac.kr (S.-Y. Nah).1 First two authors contributed equally to this work.

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2006.02.034

structure to levorphanol, codeine, and morphine, but has a lowaffinity for opiate receptors and is not considered addictive. DMhas been used as an effective antitussive drug for more than40 years. Although DM alone does not have analgesic activity,reports have suggested that it is capable of attenuating thetolerance and/or enhancing the analgesia produced by morphine(Elliott et al., 1994; Mao et al., 1996) and nonsteroidal anti-inflammatory drugs (Price et al., 1996). Moreover, recent studieshave demonstrated that DM exhibits anticonvulsant andneuroprotective effects in models of glutamate- and kainate-induced neurotoxicity (Choi, 1987; Liu et al., 2003; Kim et al.,1996, 2001a,b; Yin and Sun, 1999; Shin et al., 2004).

However, despite its beneficial effects, DM has reportedlyexhibited toxicity in children (Pender and Parks, 1991), has been

Fig. 1. Chemical structures of the dextrorotatory morphinan analogs.

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found to trigger psychotomimetic reactions (Holtzman, 1994;Katona and Wason, 1986; Kim and Jhoo, 1995), and was shownto have abuse potential when ingested at high doses by adoles-cents (Jhoo and Kim, 1990; Rammer and Hormgren, 1988). Wepreviously demonstrated that DM could produce psychotropicbehavioral patterns in an animal model when given alone, andcould potentiate behavioral effects in rodents when given in

Fig. 2. Effect of DM and its analogs DF, HM, AM and CM on IACh in oocytesexpressing α3β4 nicotinic acetylcholine receptors. (A) Acetylcholine (30 μM)was applied with or without DM, DF, HM, AM or CM (30 μM each). Co-application of DM and its analogs inhibited IACh. Tracings are representative ofsix separate oocytes from three different frogs. (B) The histograms show thepercent blockade of IACh by DM and its analogs. The resting membrane potentialof the oocytes was about −35 mV, and oocytes were voltage-clamped at aholding potential of −80 mV prior to drug application.

conjunction with cocaine (Jhoo et al., 2000; Kim et al., 1997).Moreover, we showed that chronic DM administration couldperturb cellular immune responses in a manner similar to theimmunosuppressive effects caused by phencyclidine (Kim andJhoo, 1995).

In an effort to identify a DM analog or metabolite with feweradverse effects than DM, but comparable anticonvulsant and/orneuroprotective properties, we developed a series of analogcompounds modified at positions 3 and 17 of the morphinanring system. These include 3-allyloxy-17-methoxymorphinan(AM), 3-cyclopropyl-17-methoxymorphinan (CM) and 3-hydroxymorphinan (HM) (Fig. 1) (Kim et al., 2001b). Wefurther showed that these compounds exhibited anti-convulsantactivities and negligibly weak psychotropic effects (Kim et al.,2003). It is our hope that these compounds will prove useful asanti-additive and neuroprotective agents.

The nicotinic acetylcholine receptor is one of most exten-sively investigated receptors among the ligand-gated ion chan-nels. Acetylcholine activates the nicotinic acetylcholinereceptor, allowing the influx of cations (mostly Na+ ions) intocells through the channel pore. The nicotinic acetylcholinereceptor is formed of α and/or β subunits. The α7, α9, and α10subunits can form functional homomeric receptors, whereas theother α subunits must combine with β subunits to form

Fig. 3. Time-course effect of DM on IACh in oocytes expressing α3β4 nicotinicacetylcholine receptors. (A) IACh in oocytes expressing α3β4 nicotinicacetylcholine receptors was elicited at a holding potential of −80 mV for theindicated times (1, 3, 10, 30 or 60 s) in the presence of 30 μM acetylcholine withor without 100 μM DM. Tracings are representative of eight separate oocytesfrom three different frogs. (B) The % inhibition of IACh induced by DM wascalculated from the average of the peak inward current elicited by acetylcholinealone and the peak inward current elicited by co-application of DM withacetylcholine. Each histogram represents the mean±S.E.M. (n=8/group).

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functional heteromers, with the precise composition determinedby the organ type or nervous system region (Sargent, 1993). Forexample, the muscular nicotinic receptor channel consists of theα1β1δγ (embryonic form) or α1β1δε (adult form) subunits(Lindstrom, 1995), whereas the neuronal nicotinic receptorconsists of the α (α2−α10) and β (β2−β4) subunits (Sargent,1993). DM and its metabolite, dextrorphan, inhibited α3β4nicotinic acetylcholine receptor channel activity in heterolo-gously expressed HEK293 cells (Hernandez et al., 2000),suggesting that α3β4 nicotinic acetylcholine receptors might bea main target for putative anti-addictive agents such as DM andDM analogs. However, no previous study has examined whetherthe DM analogs, HM, AM and CM, are capable of regulatingα3β4 nicotinic acetylcholine receptor channel activity.

Accordingly, we herein examined whether HM, AM and CMexerted inhibitory effects on acetylcholine-elicited inward peakcurrents (IACh) in Xenopus oocytes expressing bovine α3β4nicotinic acetylcholine receptor subunit cRNAs, a model systemthat has few endogenous ion channels (Dascal, 1987) and allowsheterologous expression of ion channels for various biochemicalstudies (Choi et al., 2002, 2003; Sala et al., 2002). We also testedwhether these effects were mediated through competition withacetylcholine binding sites, and compared our results with theinhibition induced by DM and DF. Our results revealed that DM,DF, HM, AM and CM all inhibited IACh in a dose-dependent,voltage-independent, and reversible manner. The order of potencyfor the inhibition of IACh was CM N HM N DF = AM N DM.

Fig. 4. The dose–response relationships of DM, DF and mecamylamine on IACh in ooocytes expressing α3β4 nicotinic acetylcholine receptors was elicited at a holding poand then the indicated concentration of DM, DF or mecamylamine was co-appliedcalculated from the average of the peak inward current elicited by acetylcholine alonecontinuous line shows the curve fitted according to the equation y /ymax=[DM, Dmaximum inhibition of the α3β4 receptor-mediated current, K1 / 2 is the concentconcentration of DM, DF, or mecamylamine. Each point represents the mean±S.E.M

2. Materials and methods

2.1. Materials

Fig. 1 shows the chemical structures of DM, DF, HM, AMand CM. The cDNAs for the bovine α3 and β4 subunits of thenicotinic acetylcholine receptor were kindly provided by Dr. S.Sala (Universidad Miguel Hernández-Consejo Superior deInvestigaciones Cientificas, Spain). The test compounds usedin this study were dissolved in dimethyl sulfoxide (DMSO) andwere diluted with bath medium before use. The final DMSOconcentration was less than 0.05% in each case. Otherchemical agents were obtained from Sigma (St. Louis, MO,USA).

2.2. Collection of Xenopus oocytes

The care and handling of Xenopus laevis frogs were per-formed in accordance with the guide for the Care and Use ofLaboratory Animals published by the NIH (USA) and with thehighest standards of institutional guidelines, Republic of Korea.For isolation of oocytes, frogs were anesthetized with an aeratedsolution of 3-amino benzoic acid ethyl ester and oocytes weresurgically removed. Frogs underwent surgery only twice, withsurgeries separated by at least 3 weeks. Oocytes were separatedby collagenase treatment and gently shaken for 2 h in CaCl2-freemedium containing 82.5 NaCl, 2 mMKCl, 1 mMMgCl2, 5 mM

ocytes expressing α3β4 nicotinic acetylcholine receptors. (A, B, and C) IACh intential of −80 mV for the indicated times in the presence of 30 μM acetylcholine,. (D) The % inhibition of IACh induced by DM, DF and mecamylamine wasand the peak inward current after DM, DF or mecamylamine co-application. TheF, or mecamylamine] / [DM, DF, or mecamylamine]+K1 / 2, where ymax is theration for half-maximum inhibition, and [DM, DF, or mecamylamine] is the. (n=9–12/group).

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HEPES, 2.5 mM sodium pyruvate, 100 units of penicillin per mland 100 μg streptomycin/ml. Stage 5 or 6 oocytes were collectedandmaintained at 18 °Cwith continuous gentle shaking in ND96(96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and5 mMHEPES, pH 7.5) supplemented with 0.5 mM theophyllineand 50 μg gentamycin/ml. All solutions were changed daily. Allexperiments were performed within 2–4 days of oocyteisolation.

2.3. Recording of acetylcholine-induced inward currents

For experiments, a single oocyte was placed in a smallPlexiglas net chamber (0.5 ml) and constantly superfused withND96 medium in the absence or presence of acetylcholine and/or drugs during recording. The microelectrodes were filled with3 M KCl and had resistances of 0.2–0.7 MΩ. Two-electrodevoltage-clamp recordings were performed at room temperaturewith an Oocyte Clamp (OC-725C; Warner Instrument,Hamden, CT, USA) and the Digidata 1200A. For most of theelectrophysiological experiments, the oocytes were clamped ata holding potential of −80 mV and the current–voltagerelationship was obtained using 400-ms duration voltageramps from −100 and +60 mV. Linear leak and capacitancecurrents were corrected by means of the leak subtractionprocedure.

Fig. 5. The dose–response relationship of HM-, AF- or CM-induced inhibition of IAChin oocytes expressing α3β4 nicotinic acetylcholine receptors was elicited at a hoacetylcholine, and then the indicated concentrations of HM, AF or CM were applied.average of the peak inward current elicited by acetylcholine alone and the peak inwshows the curve fitted according to the equation, y /ymax=[HM, AF, or CM] / [HM, Amediated current, K1 / 2 is the concentration that elicits half-maximum inhibition, andthe mean±S.E.M. (n=9–12/group).

2.4. In vitro RNA synthesis

Recombinant plasmids containing α3β4 cDNA inserts werelinearized by digestion with the appropriate restrictionenzymes, and cRNAs were generated with an in vitrotranscription kit (mMessage mMachine; Ambion, Austin, TX,USA) using T3 or T7 polymerase. The RNA was dissolved inRNase-free water at 1 μg/μl, divided into aliquots, and stored at−70 °C until use.

2.5. Oocyte injection

Oocytes were injected with H2O or various nicotinicacetylcholine receptor cRNAs (5–20 ng) using a NanojectAutomatic Oocyte Injector (Drummond Scientific, Broomall,PA, USA). The injection pipette was pulled from glass capillarytubing, and the tip was broken to ≈ 20-μm in diameter.

2.6. Data analysis

All values are presented as means±S.E.M. The differencesbetween means of results from oocytes treated with acetylcho-line alone or co-treated with drugs were analyzed using theunpaired Student's t test. A value of Pb0.05 was consideredstatistically significant.

in oocytes expressing α3β4 nicotinic acetylcholine receptors. (A, B, and C) IAChlding potential of −80 mV for the indicated times in the presence of 30 μM(D) The % inhibition of IACh induced by HM, AF or CM was calculated from theard current elicited after co-application of HM, AF or CM. The continuous lineF, or CM]+K1 / 2, where ymax is the maximum inhibition of the α3β4 receptor-[HM, AF, or CM] is the concentration of HM, AF, or CM. Each point represents

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3. Results

3.1. Effect of DM, DF, HM, AM or CM on IACh in oocytesexpressing α3β4 nicotinic acetylcholine receptors

The addition of acetylcholine (30 μM) to the bath solutioninduced a large inward current in oocytes injected with bovineα3β4 nicotinic acetylcholine receptor subtypes, indicating thatthe desired nicotinic acetylcholine receptors were functionallyexpressed in this system (Fig. 2A). Treatment of oocytes withDM, DF, HM, AM or CM alone had no effect in oocytesexpressing α3β4 nicotinic acetylcholine receptors, maintainedat a holding potential of −80 mV (data not shown). In contrast,co-treatment of the oocytes with DM, DF, HM, AM or CM (each30 μM) and 30 μM acetylcholine yielded a significantly lowerpeak IACh than seen in the presence of acetylcholine alone (Fig.2A and B; n=6–8 from three different frogs). This inhibition ofpeak IACh by DM, DF, HM, AM or CMwas reversible (Fig. 2A).The percent inhibition of peak IACh was 53.2±10.2%, 68.3±5.3%, 60.3±10.3%, 62.4±2.3%, and 72.0± 8.3% for DM, DF,HM, AM and CM, respectively (Fig. 2B).

3.2. Time-course effect of DM on IACh in oocytes expressingα3β4 nicotinic acetylcholine receptors

We next examined the time-dependent effects of DM on IAChin oocytes expressing α3β4 nicotinic acetylcholine receptors.As shown in Fig. 3A, the peak IACh induced by treatment with30 μM alone was almost fully saturated after 30 s treatment. Co-

Fig. 6. Current–voltage relationship and voltage-independent inhibition. (A and Bduration voltage ramps from −100 and +60 mV. Voltage steps were applied before anDF or DM (A) or the absence or presence of 100 μMAM, CM or HM (B). (C and D)potentials in oocytes expressing α3β4 nicotinic acetylcholine receptors. Oocytes werEach point represents the mean±S.E.M. (n=9–12/group).

treatment of the oocytes with acetylcholine plus 100 μM DMinhibited the peak IACh by 17.8±14.5%, 43.6±7.1%, 62.8±5.0%, 74.1±7.5%, and 81.5±1.1% after 1, 3, 10, 30, and 60 s,compared to oocytes treated with acetylcholine alone. Thesefindings indicate that the blocking effects of DM increased withincreasing application time until a steady-state inhibition levelwas achieved after 30 s (Fig. 3B).

3.3. Concentration-dependent inhibition of IACh by DM, DF,HM, AM or CM in oocytes expressing α3β4 nicotinicacetylcholine receptors

Dose–response experiments revealed that co-treatment withacetylcholine plus varying concentrations of DM, DF, HM, AMor CM dose-dependently inhibited IACh in oocytes expressingα3β4 nicotinic acetylcholine receptors (Figs. 4 and 5). The IC50

values were 19.5±5.2, 12.5±2.7, 13.5±4.0, 16.3±1.8 and 10.1±2.8 μM for DM, DF, HM, AM and CM, respectively (n=9–12from three different frogs). The Hill coefficients were 0.89±0.1,0.91±0.13, 0.87±0.15, 1.02±0.11, and 0.8±0.11 for DM, DF,HM, AM and CM, respectively. For comparison, we alsotested the effect of mecamylamine, one of non-competitivenicotinic acetylcholine receptor antagonists, on IACh, andfound that it was also dose-dependent up to 30 μM (Fig. 4Cand D), with an IC50 value of 3.2±0.9 μM. These results indicatethat DM, DF, HM, AM, CM and mecamylamine all regulateα3β4 nicotinic acetylcholine receptors, and that mecamylamineis about 3- to 5-fold more potent than DM and its analogs (Figs. 4and 5).

) The representative current–voltage relationship was obtained using 400-msd after application of 10 μM acetylcholine in the absence or presence of 100 μMDM-, DF-, HM-, AM- or CM-induced inhibitions at different membrane holdinge exposed to acetylcholine alone or acetylcholine + DM or DM analogs for 30 s.

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3.4. Current–voltage relationship and voltage-independentinhibition of IACh by DM, DF, HM, AM and CM in oocytesexpressing α3β4 nicotinic acetylcholine receptors

As shown in Fig. 6A and B, the current–voltage relationshipof the current induced by acetylcholine with voltage steps from−100 to +60 mV showed a slight inward rectification at morepositive potentials in oocytes expressing α3β4 nicotinicacetylcholine receptors. The reversal potential was near 0 mVin oocytes treated with acetylcholine alone and acetylcholine +DM and its analogs. These results indicate that acetylcholineinduces the cation current, and this is not affected by thepresence of DM or its analogs (n=9–12, from three differentfrogs). Our results further revealed that the inhibitory effects ofDM and the DM analogs on IACh in oocytes expressing α3β4nicotinic acetylcholine receptors were independent of the mem-brane holding potential (Fig. 6C and D). At membrane holdingpotentials of −120, −90, −60, and −30 mV DM inhibited IAChby 58.3±10.5%, 62.7±4.5%, 65.6±7.0%, and 67.2±8.0%,respectively (n=9–12, from three different frogs), DF inhibitedIACh by 51.7±5.5%, 55.1±9.2%, 52.5±7.3%, and 53.2±8.2%,respectively (n=9–12, from three different frogs), HM inhibited

Fig. 7. Dose–response relationship for acetylcholine with or without DM, DF,HM, AM or CM in oocytes expressing α3β4 nicotinic acetylcholine receptors.(A) IACh induced by the indicated concentrations of acetylcholine in the absenceCon (□) or presence of 100 μM DM (○) or DF (Δ). (B) IACh elicited by theindicated concentrations of acetylcholine in the absence Con (□) or presence of100 μM HM (○), AM (Δ) or CM (▭). Oocytes were voltage-clamped at aholding potential of −80 mVand exposed to acetylcholine with or without DMor DM analogs for 30 s. Each point represents the mean±S.E.M. (n=9–12/group).

IACh by 58.5±10.4%, 62.7±5.6%, 65.6±8.2%, and 57.2±7.5%,respectively (n=9–12, from three different frogs), AM inhibitedIACh by 55.3±3.5%, 51.7±8.11%, 55.2±2.0%, and 52.5±5.6%,respectively (n=9–12, from three different frogs), and CMinhibited IACh by 68.3±2.5%, 71.5±5.1%, 68.2±12.0%, and73.2±9.5%, respectively (n=9–12, from three different frogs).

3.5. Non-competitive inhibition of α3β4 nicotinic acetylcholinereceptors by DM, DF, HM, AM and CM

To begin studying the mechanism by which DM, DF, HM,AM, and CM inhibit IACh in oocytes expressing α3β4 nicotinicacetylcholine receptors, we analyzed the effect of 100 μM DM,DF, HM, AM or CM on the IACh evoked by differentacetylcholine concentrations (Fig. 7A and B). Co-treatment ofoocytes expressing α3β4 nicotinic acetylcholine receptors with100 μMDM, DF, HM, AM or CM plus different concentrationsof acetylcholine did not shift significantly the dose–responsecurve of acetylcholine to the right. The EC50 values were 63.7±4.9, 72.4±4.7, 82.5±14.5,86.2±4.6, 70.8±16.6 and 88.5±16.9 μM for DM, DF, HM, AM and CM, respectively, and theHill coefficients were 1.5, 1.7, 1.8, 1.5, 1.4 and 1.8,respectively. Thus, DM, DF, HM, AM and CM significantlyinhibited the IACh elicited by 10, 30 and 100 μM ofacetylcholine, independent of the acetylcholine concentration(n=9–12 from three different frogs; Fig. 7A and B).

4. Discussion

In the present study, we demonstrated that (1) co-treatment ofoocytes expressing bovine α3β4 nicotine acetylcholine recep-tors with acetylcholine plus DM and other dextrorotatorymorphinans (HM, AM and CM) inhibited IACh in a reversibleand dose-dependent manner, (2) co-treatment of these oocyteswith DM and acetylcholine induced a steady-state inhibitionafter 30 s, (3) the inhibition of IACh by DM, DF, HM, AM andCM occurred in a non-competitive and voltage-independentmanner in these oocytes, and (4) CM showed the most potentinhibition of IACh in these oocytes, while HM, AM and DMshowed similar potencies.

Although future work will be required to determine how DM,DF, HM, AM and CM inhibit IACh in oocytes expressing bovineα3β4 nicotinic acetylcholine receptors, one possible mechanismis that DM and its analogs may act as open channel blockers ofα3β4 nicotinic acetylcholine receptors. However, open channelblockers such as local anesthetics or hexamethonium arestrongly voltage dependent, due to charges carried in theirtransmembrane electrical fields (Sine and Taylor, 1982;Heidmann et al., 1983; Arias, 1996). As the inhibitory effectsof DM and its analogs on IACh in oocytes expressing bovineα3β4 nicotinic acetylcholine receptors were not voltage-dependent, they may not act as open channel blockers.

Another possibility is that DM and its analogs may work ascompetitive inhibitors by blocking the association of acetylcho-line with its binding site(s) on α3β4 nicotinic acetylcholinereceptors. However, our competition experiments revealed thatthe presence of DM and its analogs did not shift the

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concentration requirements of acetylcholine in oocytes expres-sing bovine α3β4 nicotinic acetylcholine receptors (Fig. 7).Thus, it appears as though DM and its analogs may act as non-competitive inhibitors of α3β4 nicotinic acetylcholinereceptors.

On the other hand, α3β4 nicotinic acetylcholine receptorsappear to play various location-dependent roles. In the gut, thesereceptors are densely expressed in the myenteric neurons andmay mediate excitatory cholinergic neurotransmission andintestinal peristalsis (Zhou et al., 2002). In the peripheralnervous systems, α3β4 nicotinic acetylcholine receptors areexpressed in adrenal chromaffin cells, where they are involved incatecholamine release (Campos-Caro et al., 1997). In the centralnervous system, the receptors are widely present in thecerebellum, dorsal tegmentum, subiculum, anteroventral tha-lamic nucleus and the locus coeruleus (Perry et al., 2002;Whiteaker et al., 2002). The nuclei of the habenulo–inter-peduncular pathway show the highest densities of á3â4 receptorscompared to other areas in the brain, and are innervated byafferent terminals of the interpeduncular nucleus. Several studieshave demonstrated that this area could be a novel target of anti-additive agents such as DM, mecamylamine, and 18-methox-ycoronaridine (Maisonneuve and Glick, 2003; Mulle et al.,1991; Quick et al., 1999; Sheffield et al., 2000).

In summary, we herein demonstrated that DM and DManalogs such as the newly synthesized HM, AM and CMinhibited IACh in oocytes expressing bovine neuronal α3β4nicotinic acetylcholine receptors. Our observation that HM, AMand CM inhibit IACh with equal or greater potency than DM andDF suggests that these analogs might serve as novel non-competitive antagonistic agents against α3β4 nicotinic acetyl-choline receptors.

Acknowledgments

This study was supported by a grant (R01-2003-000-10435-0) from the Korea Science and Engineering Foundation(KOSEF), a grant (#M103K V01000803K2201 008020) fromthe Brain Research Center and the 21st Century FrontierResearch Program, Neurobiology Research Program, Bio-Foodand Drug Research Center at Konkuk University, Chungju,funded by the Ministry of Science and Technology, Republic ofKorea, and Bio/Molecular Informatics Center (KRF2004-F00019). We thank Dr. S. Sala (Universidad Miguel Hernán-dez-Consejo Superior de Investigaciones Cientificas, Spain) forgenerously providing the bovine α3β4 nACh receptor clone.

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