European Journal of Pharmacology 601 (2008) 103–105

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European Journal of Pharmacology

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Neuropharmacology and Analgesia

The novel nicotinic receptor antagonist N,N′-dodecane-1,12-diyl-bis-3-picoliniumdibromide decreases nicotine-induced dopamine metabolism in ratnucleus accumbens

Shafiqur Rahman a,1, Nichole M. Neugebauer b, Zhenfa Zhang c, Peter A. Crooks c,Linda P. Dwoskin a,c, Michael T. Bardo a,b,⁎a Center for Drug Abuse Research Translation, University of Kentucky, Lexington, KY 40506, USAb Department of Psychology, University of Kentucky, Lexington, KY 40506, USAc Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA

⁎ Corresponding author. Center for Drug Abuse ReseKentucky, BBSRB, Room447, 741 S. Limestone, Lexington, K257 6456; fax: +1 859 257 3235.

E-mail addresses: Shafiqur.Rahman@sdstate.edu (S. R(M.T. Bardo).

1 Current address: Department of Pharmaceutical SSouth Dakota State University, Brookings, SD 57007, USA

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.10.037

a b s t r a c t

a r t i c l e i n f o

Article history:

The current study examin Received 30 July 2008Received in revised form 30 September 2008Accepted 10 October 2008Available online 26 October 2008

Keywords:bPiDDBNicotinic receptorDopamine metabolismDOPACNucleus accumbensHPLC

ed the effect of the novel nicotinic acetylcholine receptor antagonist, N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), on nicotine-induced dopamine metabolism in ratnucleus accumbens, striatum and medial prefrontal cortex. Acute nicotine (0.5 mg/kg, s.c.) produced anincrease in the content of dihydroxyphenylacetic acid (DOPAC) in nucleus accumbens, but not in striatum ormedial prefrontal cortex. Pretreatment with bPiDDB (1 or 3 mg/kg, s.c.) dose-dependently inhibited thenicotine-induced increase in DOPAC content in nucleus accumbens. These results indicate that bPiDDBinhibits the nicotine-induced increase in DOPAC in reward-relevant brain region targeting nicotinicacetylcholine receptors.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

The midbrain dopamine system is a target for the reinforcingactions of many addictive drugs, including nicotine (Balfour, 2004;Di Chiara et al., 2004; Picciotto and Corrigall, 2002). In themammalianbrain, nicotine exerts its effects by stimulating a variety of nicotinicacetylcholine receptors, which are distributed throughout the braindopamine system (Klink et al., 2001; Gotti et al., 2006). Given theabundant number of nicotinic acetylcholine receptors in the brain, it islikely that the development of nicotinic antagonists that specificallyblock nicotinic acetylcholine receptors involved in modulatingmidbrain dopamine function may offer novel pharmacotherapeuticapproaches to treat nicotine dependence.

Recently, our laboratory has synthesized a number of bis-azaaro-matic quaternary ammonium analogs as nicotinic acetylcholinereceptors antagonists (Ayers et al., 2002). The central inhibitory effectsof the novel bis-analog, N,N′-dodecane-1,12-diyl-bis-3-picolinium

arch Translation, University ofY 40536-0509, USA. Tel.: +1859

ahman), mbardo@uky.edu

ciences, College of Pharmacy,.

l rights reserved.

dibromide (bPiDDB), are realized following its entry into brain viathe blood brain barrier choline transporter (Geldenhuys et al., 2005;Lockman et al., 2008; Albayati et al., 2008). bPiDDB inhibits nicotine-evoked striatal dopamine release in vitro (Dwoskin et al., 2004, 2008)and reduces the acute nicotine-induced increase in extracellulardopamine in rat nucleus accumbens (Rahman et al., 2007, 2008),which likely explains, at least in part, the ability of bPiDDB to decreaseintravenous nicotine self-administration in rats (Neugebauer et al.,2006). bPiDDB does not inhibit dopamine uptake by the striataldopamine transporter (Dwoskin et al., 2008), and thus it does notappear to interact directly with this plasmalemma protein. However, itremains to be determined if bPiDDB alters dopamine metabolism todihydroxyphenylacetic acid (DOPAC) by monoamine oxidase. Thepresent study examined the effects of bPiDDB on the acute nicotine-induced increase in tissue content of DOPAC in rat nucleus accumbens,striatum and medial prefrontal cortex using an ex-vivo assay and highperformance liquid chromatography with electrochemical detection(HPLC-EC).

2. Materials and methods

2.1. Animal

Male Sprague–Dawley rats (250–275 g) were used. Rats had ad libaccess to food and water and were maintained on a 12:12 light/dark

Fig. 2. Effects of systemic bPiDDB (1 or 3 mg/kg) on the acute nicotine (NIC; 0.5 mg/kg)-induced changes in DOPAC content in rat striatum (upper panel) and medial prefrontalcortex (lower panel). bPiDDB was administered 15 min prior NIC and rats were killed30min after NIC administration. Data are expressed as µg/g tissue (mean±S.E.M, n=7–8rats/group).

104 S. Rahman et al. / European Journal of Pharmacology 601 (2008) 103–105

cycle (lights on at 0700 h). Experimental protocols were in accordancewith the NIH Guide for the Care and Use of Laboratory Animals andwereapproved by the Institutional Animal Care and Use Committee at theUniversity of Kentucky.

2.2. Drugs and dosage regimen

S(−)-Nicotine ditartrate was prepared in saline solution. Doses ofnicotine represent the free base weight. Both bPiDDB and mecamy-lamine HCl were prepared in saline and doses represent the saltweights. All drugs were administered subcutaneously (sc).

2.3. Brain tissue preparation and HPLC analysis of DOPAC content

Ratswere randomlyassigned tooneof six treatmentgroups: (1) saline;(2) nicotine (0.5 mg/kg) alone; (3) bPiDDB 1 mg/kg alone; (4) bPiDDB3 mg/kg alone; (5) bPiDDB 1 mg/kg plus nicotine (0.5 mg/kg) and(6) bPiDDB 3 mg/kg plus nicotine (0.5 mg/kg). In a separate study, theeffect of the nonselective nicotinic acetylcholine receptor antagonistmecamylamine (5 mg/kg) on nicotine-induced changes in DOPAC wasdetermined. Each group consisted of 8–10 rats. Doses of antagonist weregiven15minbeforenicotine (0.5mg/kg) administration.Dosesof nicotine,bPiDDB and mecamylamine were selected based on previous reports(Brazell et al., 1990; Janhunen et al., 2005; Neugebauer et al., 2006;Rahmanet al., 2007). Thirtyminafter nicotineor saline injection, ratswerekilled by rapid decapitation, brains were obtained, and nucleus accum-bens, medial prefrontal cortex and striatum dissected on an ice-colddissection plate as described previously (Zhu et al., 2004). Brain regionsfrom individual rats were stored at −70 °C in 10 volumes (medialprefrontal cortex) or 20 volumes (nucleus accumbens and striatum) of0.1 N perchloric acid until assay. Upon assay, samples were thawed on ice,sonicated and centrifuged at 30,000 ×g for 15min at 4 °C. For each sample,20 μl of the resulting supernatant was injected onto a high performanceliquid chromatography system coupled with electrochemical detectionunit (HPLC-ECD, ESA Inc., Chelmsford, MA) to determine DOPAC content.DOPAC was measured because it represents the major dopaminemetabolite in rat ventral striatum (see Wilk and Stanley, 1978). TheHPLC system consisted of a solvent delivery unit, a Coulochem IIIelectrochemical detector equippedwith a 5011analytical cell and a 5020guard cell. The guard cell was set at +350 mV, electrode 1 at −150 mV,and electrode 2 at +200 mV. The mobile phase consisted of 75 mMNaH2PO4, 1.7 mM 1-octanesulfonic acid, 25 μM EDTA, 100 μl/Ltriethylamine and 10% acetonitrile (adjusted to pH 3.0 with phosphoricacid), and theflow ratewas0.5ml/min. Sampleswere loaded into a 20 μlsample loop and injected onto a reverse phase analytical column(BetaBasic-18 column, 150mm×3mm, Thermo Hypersil-Keystone, PA).Chromatograms were integrated, compared with the standards andanalyzed using an ESA Chromatography Data System (EZChrom Elite,

Fig. 1. Effects of systemic bPiDDB (1 or 3 mg/kg) on the acute nicotine (NIC; 0.5 mg/kg)-induced increase in DOPAC content in nucleus accumbens. bPiDDB was administered15 min prior NIC and rats were killed 30 min after NIC administration. Data areexpressed as µg/g tissue (mean±S.E.M, n=7–8 rats/group). ⁎Pb0.05, indicatessignificant difference from saline control (SAL+SAL), # Pb0.05, indicates significantdifference from NIC alone (SAL+NIC 0.5 mg/kg).

Chelmsford, MA). Peak heights and calibration factors based onstandards were used to calculate the amount of DOPAC.

2.4. Data analysis

The amount of tissue DOPAC level was expressed as µg/g tissuewetweight and analyzed for significant differences by one way analysis ofvariance (ANOVA) followed by post hoc tests for multiple comparison.The significance level was set at Pb0.05.

3. Results

A oneway ANOVA revealed a significant main effect of treatment, F(5, 41)=2.83, Pb0.05, in nucleus accumbens (Fig. 1), but not in eitherstriatum or medial prefrontal cortex (Fig. 2). Post-hoc tests revealedthat nicotine (0.5mg/kg, s.c.) produced a significant increase in DOPACcontent in nucleus accumbens. There was also a significant differencebetween the bPiDDB (3 mg/kg) plus nicotine and the nicotine alonegroups in nucleus accumbens DOPAC. Pretreatment with bPiDDB (1 or3mg/kg) dose-dependently inhibited the nicotine-induced increase inDOPAC content in nucleus accumbens. Importantly, rats treated withbPiDDB (3 mg/kg) and nicotine did not differ significantly from salinecontrol, indicating that the inhibition of nicotine-induced dopaminemetabolism was complete. Similarly, pretreatment with mecamyla-mine (5 mg/kg, s.c.) blocked completely the nicotine-induced increasein DOPAC content in nucleus accumbens (245% vs 123% of salinecontrol; results not shown).

4. Discussion

Acute nicotine administration preferentially increased tissue DOPACcontent in nucleus accumbens, but not in striatum or medial prefrontalcortex. Pretreatment with mecamylamine, a non-selective nicotinicacetylcholine receptor antagonist, blocked the nicotine-inducedenhancement of DOPAC content, demonstrating the involvement of anicotinic acetylcholine receptor-mediated mechanism. Like mecamyla-mine, bPiDDB dose-dependently and completely inhibited the nicotine-induced increase in DOPAC content in nucleus accumbens. The present

105S. Rahman et al. / European Journal of Pharmacology 601 (2008) 103–105

results showing that acute nicotine preferentially elevated DOPACcontent in nucleus accumbens is consistent with previous neurochem-ical studies using brain tissue assays and microdialysis procedures(Benwell and Balfour, 1992; Brazell et al., 1990; Di Chiara and Imperato,1988; Janhunen et al., 2005; Mitchell et al., 1989; Nissell et al., 1994).Moreover, the blockade of the nicotine-induced increase in DOPACcontent produced by pretreatmentwithmecamylamine is also in accordwith previous tissue dopamine metabolism studies (Brazell et al., 1990;Mitchell et al., 1989; Nissell et al., 1994). Overall, these results indicatethat acute nicotine produces a specific effect on tissue DOPAC content inrat nucleus accumbens and that this effect is mediated by nicotinicacetylcholine receptors.

Pretreatment with bPiDDB dose-dependently inhibited the nico-tine-induced increase in DOPAC content in nucleus accumbens,suggesting that bPiDDB acts similarly to mecamylamine in regulatingdopamine metabolism in nucleus accumbens. However, evidencesuggests that bPiDDB may be more selective than mecamylamine inblocking a subset of mecamylamine-sensitive nicotinic acetylcholinereceptors (i.e.,α6β2-containing) in the brain (Dwoskin et al., 2008). Inany case, the ability of bPiDDB to inhibit the effect of nicotine ondopamine metabolism observed in this report extends our previousmicrodialysis studies showing that bPiDDB also inhibits nicotine-induced dopamine release in nucleus accumbens (Rahman et al., 2007,2008). The observation that acute nicotine did not have an effect onDOPAC content in the striatum or medial prefrontal cortex in thisstudy remains to be explained. Regional variations in nicotine-inducedeffects on dopamine metabolism and function have been reportedpreviously in both in vivo and in vitro studies (Brazell et al., 1990;Janhunen et al., 2005; Mitchell et al., 1989). Several factors mayaccount for the region-specific effects of acute nicotine, includingdifferential distribution of pre-synaptic nicotinic acetylcholine recep-tors and/or dopamine transporter function in specific brain areas. Forexample, acute nicotine has been shown to increase dopaminetransporter function in rat striatum and prefrontal cortex through atrafficking-independent mechanism and trafficking-dependentmechanism, respectively (Middleton et al., 2007; Zhu et al., 2007).Whether regional differences in dopamine transporter function andregulation are contributing to the differential effects of nicotine onDOPAC content in the current report remains to be determined.Nevertheless, the ability of mecamylamine and bPiDDB to block thenicotine-induced increase in DOPAC in nucleus accumbens indicatesthat dopamine utilization in this critical reward-relevant region isregulated by nicotinic acetylcholine receptors.

Acknowledgements

This work was supported by USPHS grant U19 DA17548. Forpurposes of full disclosure, the University of Kentucky holds patentson bPiDDB and a potential royalty stream to L.P.D. and P.A.C. may occurconsistent with University of Kentucky policy.

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