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Direct Agonist Activity of Tricyclic Antidepressants at Distinct Opioid Receptor Subtypes Pierluigi Onali, Simona Dedoni, and Maria C. Olianas Department of Neurosciences, Section of Biochemical Pharmacology, University of Cagliari, Cagliari, Italy Received August 4, 2009; accepted October 13, 2009 ABSTRACT Tricyclic antidepressants (TCAs) have been reported to interact with the opioid system, but their pharmacological activity at opioid receptors has not yet been elucidated. In the present study, we investigated the actions of amoxapine, amitriptyline, nortriptyline, desipramine, and imipramine at distinct cloned and native opioid receptors. In Chinese hamster ovary (CHO) cells expressing -opioid receptors (CHO/DOR), TCAs dis- placed [3H]naltrindole binding and stimulated guanosine 5-O- (3-[ 35 S]thio)triphosphate ([ 35 S]GTPS) binding at micromolar concentrations with amoxapine displaying the highest potency and efficacy. Amoxapine and amitriptyline inhibited cyclic AMP formation and induced the phosphorylation of signaling mole- cules along the extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol-3 kinase pathways. Amox- apine also activated -opioid receptors in rat dorsal striatum and nucleus accumbens and human frontal cortex. In CHO cells expressing -opioid receptors (CHO/KOR), TCAs, but not amoxapine, exhibited higher receptor affinity and more potent stimulation of [ 35 S]GTPS binding than in CHO/DOR and effec- tively inhibited cyclic AMP accumulation. Amitriptyline regu- lated ERK1/2 phosphorylation and activity in CHO/KOR and C6 glioma cells endogenously expressing -opioid receptors, and this effect was attenuated by the -opioid antagonist nor-bin- altorphimine. In rat nucleus accumbens, amitriptyline slightly inhibited adenylyl cyclase activity and counteracted the inhib- itory effect of the full agonist trans-()-3,4dichloro-N-methyl- N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488). At the cloned -opioid receptor, TCAs showed low affinity and no significant agonist activity. These results show that TCAs differ- entially regulate opioid receptors with a preferential agonist activ- ity on either or subtypes and suggest that this property may contribute to their therapeutic and/or side effects. Although the inhibition of presynaptic reuptake of mono- amines is considered to be the primary mechanism of action of tricyclic antidepressants (TCAs), it is well established that these drugs can act on multiple molecular targets by affect- ing the activity of distinct neurotransmitter receptor systems and ion channels (Baldessarini, 2006). These secondary ac- tions have been generally related to TCAs’ adverse side ef- fects, although some of them have been proposed to contrib- ute to the therapeutic activity. An interaction with the opioid system has long been shown to be involved in the analgesic and mood-elevating effects of TCAs. A number of studies have reported that the antinoci- ceptive effects of TCAs are reversed by opioid receptor antag- onists (Biegon and Samuel, 1980; Gray et al., 1998; March- and et al., 2003; Benbouzid et al., 2008a,b) and that TCAs potentiate morphine-induced analgesia both in animals (Hamon et al., 1987) and in humans (Mico ´ et al., 2006). In animal behavioral tests predictive of antidepressant effects in humans, such as the forced swimming and learned help- lessness tests, the effects of TCAs have been found to be antagonized by blockade of opioid receptors, indicating the possible participation of opioid neurotransmission in the an- tidepressant activity of these drugs (Devoize et al., 1982; Tejedor-Real et al., 1995; Besson et al., 1999). Although these studies implicate opioid mechanisms in the pharmacological activity of TCAs, the mode of action of these drugs on the opioid system has not been fully clarified. Some studies have reported that long-term antidepressant use in- creases enkephalin levels in different brain areas (De Felipe This work was supported by a grant from the Ministry of Education, University, and Research of Italy. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.159939. ABBREVIATIONS: TCA, tricyclic antidepressant; CHO, Chinese hamster ovary; [ 35 S]GTPS, guanosine 5-O-(3-[ 35 S]thio)triphosphate; NTI, naltrindole; FSK, forskolin; ()-U50,488, trans-()-3,4dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide; nor-BNI, nor-binaltor- phimine dihydrochloride; CTAP, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH 2 ; DPDPE, (2-D-penicillamine, 5-D-penicillamine)-enkephalin; DAMGO, D-Ala 2 -N-methyl-Phe-Gly-ol 5 )-enkephalin; pGSK, phospho-Ser9-glycogen synthase kinase-3; pS6rp, phospho-Ser235/236-S6 ribosomal pro- tein; ERK, extracellular signal-regulated kinase; pERK1/2, phosphorylation of extracellular signal-regulated kinase 1/2; GSK, glycogen synthase; pAkt, phospho-Thr308-protein kinase B/Akt; CHO/DOR, /KOR, /MOR, CHO cells expressing the human -, -, and -opioid receptor, respectively; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ECL, enhanced chemiluminescence; ANOVA, analysis of variance. 0022-3565/10/3321-255–265$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 332, No. 1 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 159939/3544493 JPET 332:255–265, 2010 Printed in U.S.A. 255 at ASPET Journals on June 11, 2018 jpet.aspetjournals.org Downloaded from

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Direct Agonist Activity of Tricyclic Antidepressants at DistinctOpioid Receptor Subtypes

Pierluigi Onali, Simona Dedoni, and Maria C. OlianasDepartment of Neurosciences, Section of Biochemical Pharmacology, University of Cagliari, Cagliari, Italy

Received August 4, 2009; accepted October 13, 2009

ABSTRACTTricyclic antidepressants (TCAs) have been reported to interactwith the opioid system, but their pharmacological activity atopioid receptors has not yet been elucidated. In the presentstudy, we investigated the actions of amoxapine, amitriptyline,nortriptyline, desipramine, and imipramine at distinct clonedand native opioid receptors. In Chinese hamster ovary (CHO)cells expressing �-opioid receptors (CHO/DOR), TCAs dis-placed [3H]naltrindole binding and stimulated guanosine 5�-O-(3-[35S]thio)triphosphate ([35S]GTP�S) binding at micromolarconcentrations with amoxapine displaying the highest potencyand efficacy. Amoxapine and amitriptyline inhibited cyclic AMPformation and induced the phosphorylation of signaling mole-cules along the extracellular signal-regulated kinase 1/2(ERK1/2) and phosphatidylinositol-3 kinase pathways. Amox-apine also activated �-opioid receptors in rat dorsal striatumand nucleus accumbens and human frontal cortex. In CHO

cells expressing �-opioid receptors (CHO/KOR), TCAs, but notamoxapine, exhibited higher receptor affinity and more potentstimulation of [35S]GTP�S binding than in CHO/DOR and effec-tively inhibited cyclic AMP accumulation. Amitriptyline regu-lated ERK1/2 phosphorylation and activity in CHO/KOR and C6glioma cells endogenously expressing �-opioid receptors, andthis effect was attenuated by the �-opioid antagonist nor-bin-altorphimine. In rat nucleus accumbens, amitriptyline slightlyinhibited adenylyl cyclase activity and counteracted the inhib-itory effect of the full � agonist trans-(�)-3,4dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488). Atthe cloned �-opioid receptor, TCAs showed low affinity and nosignificant agonist activity. These results show that TCAs differ-entially regulate opioid receptors with a preferential agonist activ-ity on either � or � subtypes and suggest that this property maycontribute to their therapeutic and/or side effects.

Although the inhibition of presynaptic reuptake of mono-amines is considered to be the primary mechanism of actionof tricyclic antidepressants (TCAs), it is well established thatthese drugs can act on multiple molecular targets by affect-ing the activity of distinct neurotransmitter receptor systemsand ion channels (Baldessarini, 2006). These secondary ac-tions have been generally related to TCAs’ adverse side ef-fects, although some of them have been proposed to contrib-ute to the therapeutic activity.

An interaction with the opioid system has long been shownto be involved in the analgesic and mood-elevating effects ofTCAs. A number of studies have reported that the antinoci-

ceptive effects of TCAs are reversed by opioid receptor antag-onists (Biegon and Samuel, 1980; Gray et al., 1998; March-and et al., 2003; Benbouzid et al., 2008a,b) and that TCAspotentiate morphine-induced analgesia both in animals(Hamon et al., 1987) and in humans (Mico et al., 2006). Inanimal behavioral tests predictive of antidepressant effectsin humans, such as the forced swimming and learned help-lessness tests, the effects of TCAs have been found to beantagonized by blockade of opioid receptors, indicating thepossible participation of opioid neurotransmission in the an-tidepressant activity of these drugs (Devoize et al., 1982;Tejedor-Real et al., 1995; Besson et al., 1999).

Although these studies implicate opioid mechanisms in thepharmacological activity of TCAs, the mode of action of thesedrugs on the opioid system has not been fully clarified. Somestudies have reported that long-term antidepressant use in-creases enkephalin levels in different brain areas (De Felipe

This work was supported by a grant from the Ministry of Education,University, and Research of Italy.

Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.

doi:10.1124/jpet.109.159939.

ABBREVIATIONS: TCA, tricyclic antidepressant; CHO, Chinese hamster ovary; [35S]GTP�S, guanosine 5�-O-(3-[35S]thio)triphosphate; NTI,naltrindole; FSK, forskolin; (�)-U50,488, trans-(�)-3,4dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide; nor-BNI, nor-binaltor-phimine dihydrochloride; CTAP, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2; DPDPE, (2-D-penicillamine, 5-D-penicillamine)-enkephalin; DAMGO,D-Ala2-N-methyl-Phe-Gly-ol5)-enkephalin; pGSK, phospho-Ser9-glycogen synthase kinase-3�; pS6rp, phospho-Ser235/236-S6 ribosomal pro-tein; ERK, extracellular signal-regulated kinase; pERK1/2, phosphorylation of extracellular signal-regulated kinase 1/2; GSK, glycogen synthase;pAkt, phospho-Thr308-protein kinase B/Akt; CHO/DOR, /KOR, /MOR, CHO cells expressing the human �-, �-, and �-opioid receptor, respectively;PBS, phosphate-buffered saline; BSA, bovine serum albumin; ECL, enhanced chemiluminescence; ANOVA, analysis of variance.

0022-3565/10/3321-255–265$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 332, No. 1Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 159939/3544493JPET 332:255–265, 2010 Printed in U.S.A.

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et al., 1985; Hamon et al., 1987), whereas others observed nochange or a decrease (Herman et al., 1986; Kurumaji et al.,1988). Radioligand binding studies examining the interac-tions of TCAs with opioid receptors also yielded contradictoryresults. For example, in vivo administration of TCAs hasbeen found to cause a decrease, an increase, and no change inopioid receptor density in different brain areas (Stengaard-Pedersen and Schou, 1986; Hamon et al., 1987; Chen andLawrence, 2004). Moreover, TCAs have been reported to beeither devoid of activity at brain opioid receptors (Hall andOgren, 1981) or capable of inhibiting opioid receptor bindingat pharmacologically relevant concentrations (Biegon andSamuel, 1980; Isenberg and Cicero, 1984; Wahlstrom et al.,1994). Thus, whether these drugs can directly act on opioidreceptors and whether the distinct opioid receptor subtypesare differentially affected by TCAs still remain unresolvedissues.

In the present study, we investigated the action of theTCAs amoxapine, amitriptyline, nortriptyline, desipramine,and imipramine at cloned human �-, �-, and �-opioid recep-tors individually expressed in Chinese hamster ovary (CHO)cells. Moreover, the effects of some of these drugs at opioidreceptors endogenously expressed in C6 glioma cells, ratdorsal striatum and nucleus accumbens, and postmortemhuman frontal cortex were also examined.

Materials and MethodsMaterials. [�-32P]ATP (30–40 Ci/mmol), [2,8-3H]cyclic AMP (25

Ci/mmol), [8-14C]cyclic AMP (45.1 mCi/mmol), [2,8-3H]adenine(28.8 Ci/mmol), guanosine 5�-O-(3-[35S]thio)triphosphate([35S]GTP�S) (1306 Ci/mmol), [15,16-3H]diprenorphine (53 Ci/mmol), and [5�,7�-3H]naltrindole ([3H]NTI) (20 Ci/mmol) were ob-tained from PerkinElmer Life and Analytical Sciences (Waltham,MA). Forskolin (FSK) and GTP�S were from Calbiochem (San Diego,CA) and Boehringer Ingelheim GmbH (Ingelheim, Germany), re-spectively. (�)-U50,488 hydrochloride, nor-binaltorphimine dihy-drochloride (nor-BNI), D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), and NTI hydrochloride were from Tocris Bioscience(Bristol, UK). (2-D-Penicillamine, 5-D-penicillamine)-Enkephalin(DPDPE) was purchased from Bachem AG (Bubendorf, Switzer-land). Amoxapine, amitriptyline, desipramine, nortriptyline,imipramine, and (D-Ala2-N-methyl-Phe-Gly-ol5)-enkephalin(DAMGO) were from Sigma-Aldrich (St. Louis, MO). Primaryantibodies were as follows: rabbit polyclonal to phospho-Ser9-glycogen synthase kinase-3� (pGSK), rabbit monoclonal to phos-pho-Ser235/236-S6 ribosomal protein (pS6rp), and rabbit polyclonalanti-extracellular signal-regulated kinase 1/2 (ERK1/2) from CellSignaling Technology Inc. (Danvers, MA); rabbit polyclonal anti-phospho-ERK1/2 (pERK1/2) from Neuromics (Northfield, MN); rab-bit polyclonal anti-GSK-3� (GSK) from Santa Cruz Biotechnology,Inc. (Santa Cruz, CA); rabbit polyclonal antibody to phospho-Thr308-protein kinase B/Akt (pAkt) from GenWay (San Diego, CA); andrabbit polyclonal anti-actin from Sigma-Aldrich. Horseradish perox-idase-conjugated goat anti-rabbit IgG and prestained protein stan-dards were from Santa Cruz Biotechnology, Inc. Unless otherwisespecified, the other reagents were from Sigma-Aldrich.

Cell Culture. CHO-K1 cells (American Type Culture Collection,Manassas, VA) were grown as a monolayer culture in tissue cultureflasks that were incubated at 37°C in a humidified atmosphere (5%CO2) in Ham’s F-12 medium (Invitrogen, Carlsbad, CA) containingL-glutamine and sodium bicarbonate and supplemented with 10%fetal calf serum (Invitrogen) and 0.5% penicillin/streptomycin.CHO-K1 cells stably expressing the human �-opioid receptor (CHO/DOR), �-opioid (CHO/KOR), and �-opioid receptor-1 (CHO/MOR-1)

were generated as described previously (Olianas et al., 2006). CHOcells stably expressing the human muscarinic M2 (CHO/M2) and M4

(CHO/M4) were grown as previously reported (Olianas et al., 1999).CHO/DOR cells were maintained in the presence of 350 �g/ml hy-gromycin (Invitrogen), whereas for the other recombinant cell linesthe growth medium contained 400 �g/ml geneticin (Invitrogen). C6rat glioma cells (European Collection of Cell Cultures, Porton Down,Wiltshire, UK) were grown in Ham’s F-12 medium supplementedwith 2 mM L-glutamine, 0.5% penicillin/streptomycin, and 10% fetalcalf serum in a humidified 95% air and 5% CO2 at 37°C.

Cell Membrane Preparation. Cells were washed with ice-coldphosphate-buffered saline (PBS), pH 7.4; scraped into an ice-coldbuffer containing 10 mM HEPES/NaOH, pH 7.4, and 1 mM EDTA;and lysed with a Dounce tissue grinder. The cell lysate was centri-fuged at 1000g for 2 min at 4°C. The supernatant was collected andcentrifuged at 32,000g for 20 min at 4°C. The pellet was resuspendedin homogenization buffer at a protein concentration of 1.0 to 1.5mg/ml and stored in aliquots at �80°C.

Cell Treatments and Cell Extract Preparation. Cells wereserum-starved for 24 h (CHO cells) or 48 h (C6 glioma cells) and thenexposed to the test agents for the indicated time periods, washedwith ice-cold PBS, and lysed by scraping into PBS containing 0.1%SDS, 1% Nonidet P40, 0.5% sodium deoxycholate, 2 mM EDTA, 2mM EGTA, 4 mM sodium pyrophosphate, 2 mM sodium orthovana-date, 10 mM sodium fluoride, 20 nM okadaic acid, 0.1% phosphataseinhibitor mixture 1, and 1% protease inhibitor mixture. The sampleswere sonicated for 5 s in an ice bath and stored at �80°C. Aliquots ofcell extracts were taken for protein determination. Protein contentwas determined by the method of Bradford (1976) using bovineserum albumin (BSA) as a standard.

Dissection of Brain Regions and Membrane Preparation.Male Sprague-Dawley rats (200–300 g) were used. Animals weremaintained in a 12-h light/dark cycle with food and water ad libitum.Experiments were performed in accordance with the European Com-munities Council Directive of November 24, 1986 (86/609/EEC) andwith the principles of Laboratory Animal Care in Italy (D.L. 116/92).Rats were killed by decapitation, and the dorsal striatum and nu-cleus accumbens were rapidly microdissected from 300-�m braincoronal slices as described previously (Olianas et al., 2006). Land-marks used for the dissection of the nucleus accumbens were theanterior commissure, the olfactory and lateral ventricles, the corpusstriatum, the septal nuclei, and the olfactory tubercle.

Human frontal cortex tissue was obtained at autopsy from threemale subjects (age 35–68 years, mean 56 years) within 26 to 36 hafter death. The subjects had no history of neurological or psychiatricdisease. The tissue was immediately frozen at �80°C and stored inliquid nitrogen. The specimens were provided by Professor R. Ambu(Department of Cytomorphology, University of Cagliari, Italy). Thestudy was approved by the local ethics committee.

Freshly dissected or frozen tissue samples were homogenized in anice-cold buffer containing 10 mM HEPES-NaOH, 1 mM EGTA, and 1mM MgCl2, pH 7.40, using a Teflon-glass tissue grinder (Kontes,Vineland, NJ). The homogenate was centrifuged at 27,000g for 20min at 4°C. The pellet was resuspended in the same buffer at aprotein concentration of 0.8 to 1.0 mg/ml and used immediately foradenylyl cyclase assays or stored at �80°C for binding assays.

Assay of [35S]GTP�S Binding. The binding of [35S]GTP�S wasassayed in a reaction mixture (final volume 100 �l) containing 25mM HEPES/NaOH, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 150 mMKCl, 10 kallikrein inhibitor units of aprotinin, and 1.0 nM[35S]GTP�S. The GDP concentration was optimized for each receptorsystem and was 30 �M for CHO/KOR and CHO/DOR, 10 �M forCHO/MOR-1, and 50 �M for rat and human brain membranes.Membranes (2–4 �g of protein) were preincubated for 20 min at 30°Cwith the test compounds. For each compound, control samples re-ceived an equal volume (10 �l) of vehicle. The reaction was started bythe addition of [35S]GTP�S and continued for 40 min at 30°C. Theincubation was terminated by the addition of 5 ml of ice-cold buffer

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containing 10 mM HEPES/NaOH, pH 7.4, and 1.0 mM MgCl2, im-mediately followed by rapid filtration on glass fiber filters (GF/C;Whatman International Ltd, Maidstone, UK). The filters werewashed twice with 5 ml of buffer, and the radioactivity trapped wasdetermined by liquid scintillation spectrometry. Nonspecific bindingwas determined in the presence of 50 �M GTP�S. Assays wereperformed in duplicate.

Receptor Binding Assays. Receptor binding assays were carriedout by using [3H]diprenorphine to label �- and �-opioid receptors and[3H]NTI to label �-opioid receptors and by incubating the membranepreparations at 30°C for 120 min in a buffer containing 25 mMHEPES/NaOH, pH 7.4, 10 mM MgCl2, 1 mM EDTA, and 150 mMKCl. For saturation binding assays, the concentrations of [3H]di-prenorphine and [3H]NTI ranged from 40 pM to 3 nM and from 20pM to 2 nM, respectively. For competition binding assays, the con-centration of either [3H]diprenorphine or [3H]NTI was 0.20 nM.Nonspecific binding was determined in the presence of 10 �M nal-oxone and corresponded to 4 to 12% and 12 to 30% of total [3H]di-prenorphine and [3H]NTI binding, respectively. Triplicate determi-nations were made for each experiment. Reactions were terminatedby filtration through Whatman GF/C filters presoaked with 0.1%polyethylenimine, which were washed three times with 5 ml ofice-cold buffer containing 10 mM HEPES/NaOH, pH 7.4, and 1 mMMgCl2. The radioactivity trapped was determined by liquid scintil-lation spectrometry.

Assay of [3H]Cyclic AMP Accumulation. CHO cells grown in36-mm plastic dishes were incubated in Ham’s F-12 medium con-taining 10 �Ci/ml [3H]adenine for 1 h at 37°C in an CO2 incubator.Thereafter, the medium was removed, and the cells were incubatedin an oxygenated Krebs-HEPES buffer containing 1 mM 3-isobutyl-1-methylxanthine for 10 min at 37°C. FSK (10 �M) and the varioustest compounds were then added, and the incubation was continuedfor 10 min. Control samples were incubated in the presence of anequal volume of vehicle. The incubation was stopped by the aspira-tion of the medium and the addition of an ice-cold solution containing6% (w/v) perchloric acid and 0.1 mM [14C]cyclic AMP (4000 cpm).After 30 min at ice-bath temperature, the solution was neutralizedby the addition of ice-cold 0.6 M KOH and left on ice for an additional30 min. After centrifugation at 20,000g for 5 min, the supernatantwas collected, and [3H]cyclic AMP was isolated by sequential chro-matography on Dowex (Bio-Rad Laboratories, Hercules, CA) andalumina columns. The recovery of [3H]cyclic AMP from each samplewas corrected based on the recovery of [14C]cyclic AMP.

Assay of Adenylyl Cyclase Activity. The adenylyl cyclase ac-tivity was assayed in a reaction mixture (final volume 100 �l) con-taining 50 mM HEPES/NaOH, pH 7.4, 2.3 mM MgCl2, 0.3 mMEGTA, 0.05 mM [�-32P]ATP (150 cpm/pmol), 0.5 mM [3H]cyclic AMP(80 cpm/nmol), 1 mM 3-isobutyl-1-methylxanthine, 5 mM phospho-creatine, 50 U/ml creatine phosphokinase, 100 �M GTP, 50 �g ofBSA, 10 �g of bacitracin, and 10 kallikrein inhibitor units of apro-tinin. FSK was present at the final concentration of 10 �M. Thereaction was started by the addition of the tissue preparation (20–25�g of protein) and was carried out at 25°C for 20 min. The reactionwas stopped by the addition of 200 �l of a solution containing 2%(w/v) SDS, 45 mM ATP, and 1.3 mM cyclic AMP, pH 7.5. Cyclic AMPwas isolated by sequential chromatography on Dowex (Bio-Rad Lab-oratories) and alumina columns. The recovery of [32P]cyclic AMPfrom each sample was calculated based on the recovery of [3H]cyclicAMP. Assays were carried out in duplicate.

Western Blot Analysis. Aliquots of cell extracts containing equalamount of protein were subjected to SDS-polyacrylamide gel electro-phoresis, and the proteins were electrophoretically transferred topolyvinylidene difluoride membranes (Hybond-P; GE Healthcare,Piscataway, NJ). The efficiency of the transfer was controlled by gelstaining and by following the transfer of prestained protein stan-dards. Nonspecific binding sites were blocked by incubation in 20mM Tris-HCl, 137 mM NaCl, and 0.1% Tween 20, pH 7.6, containing5% BSA for 1 h. After washing with Tris-buffered saline containing

Tween 20, the membranes were incubated overnight at 4°C with oneof the following primary antibodies: pERK1/2 (1:10,000); ERK1/2(1:1000), pGSK-3� (1:1000), GSK (1:1000), pAkt (1:1000), or actin(1:1000). The membranes were then incubated with a horseradishperoxidase-conjugated secondary antibody (1:10,000), and immuno-reactive bands were detected by using enhanced chemiluminescence(ECL) Plus and ECL Hyperfilm (GE Healthcare). The size of theimmunoreactive bands was determined by using molecular weightstandards detected with a specific antibody suitable for the ECLsystem (Santa Cruz Biotechnology, Inc.). Band densities were deter-mined by densitometric analysis using Image Scanner III (GEHealthcare) and National Institutes of Health ImageJ software (Be-thesda, MD). The optical density of phosphoprotein bands was nor-malized to the density of the corresponding total protein or actinband to yield the relative optical density value.

Assay of ERK1/2 Activity. ERK1/2 activity was measured by anonradioactive kinase assay kit according to the manufacturer’sinstructions (Cell Signaling Technology Inc.). In brief, serum-starvedC6 glioma cells were treated with the test drugs for the indicatedtime periods, washed with ice-cold PBS, and incubated for 5 min withice-cold cell lysis buffer (25 mM Tris-HCl, pH 7.50, 150 mM NaCl, 1mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate,1 mM �-glycerolphosphate, 1 mM sodium orthovanadate, and 1�g/ml leupeptin). An aliquot of each cell extract was incubated over-night with immobilized antibody against phospho-ERK1/2 with gen-tle rocking at 4°C. After immunoprecipitation, the pellets were sus-pended in kinase buffer (25 mM Tris-HCl, pH 7.50, 5 mM�-glycerolphosphate, 0.1 mM sodium orthovanadate, 2 mM dithio-threitol, and 10 mM MgCl2) supplemented with 200 �M ATP and 40�g/ml Elk-1 fusion protein as a substrate and incubated for 30 min at30°C. The samples were then subjected to SDS-polyacrylamide gelelectrophoresis and Western blotting using a rabbit polyclonal anti-phospho-Elk-1 (Ser383) (pElk-1) antibody (1:1000). For each sample,an aliquot of the corresponding cell extract was analyzed for totalERK1/2 protein, and the optical density of the pElk-1 band wasnormalized to the density of the ERK1/2 bands.

Statistical Analysis. Results are reported as mean S.E.M.Data from concentration-response curves were analyzed usingGraphPad Software Inc. Prism (San Diego, CA), which yielded ago-nist concentration producing half-maximal effect (EC50 values) andmaximal effects (Emax). The percentage of maximal effect (% Emax) bythe drugs at �- and �-opioid receptors was calculated as Emax of theagonist/Emax of the reference full agonist (DPDPE or U50,488, re-spectively) � 100. Saturation binding data were analyzed by thenonlinear curve-fitting program LIGAND, which provided liganddissociation constant (KD) and maximal binding capacity (Bmax).Antagonist potencies were analyzed by nonlinear regression analy-sis. When increasing concentrations of antagonists in the presence ofa fixed concentration of agonist were examined, the antagonist in-hibitory constant (Ki) was calculated according to the equation: Ki �IC50/1 (A/EC50), where IC50 is the antagonist concentration pro-ducing half-maximal inhibition, A is the agonist concentration, andEC50 is the agonist EC50 value. When the effect of a fixed drugconcentration in the presence of increasing agonist concentrationswas examined, the Ki value was calculated according to the equation:EC50b � EC50a (1 I/Ki), where EC50a and EC50b are agonist EC50

values in the absence and in the presence of the antagonist, respec-tively, and I is the concentration of the antagonist. Statistical anal-ysis was performed by either Student’s t test when comparing twogroups or one-way analysis of variance (ANOVA) followed by eitherDunnett’s or Newman-Keuls post hoc tests when comparing morethan two groups.

ResultsEffects of TCAs in CHO/DOR. In CHO/DOR mem-

branes, TCAs induced a complete displacement of [3H]NTI-specific binding (Fig. 1A). The dibenzoxazepine amoxapine

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was the most potent with a Ki value (2.3 �M) approximately12- to 20-fold lower than that of the other drugs (Table 1).Scatchard analysis of saturation binding data indicated thatamoxapine (10 �M) increased the KD of [3H]NTI from 55 5to 103 9 pM (p � 0.05) without affecting the Bmax value(1470 50 versus 1430 45 fmol/mg protein) (Fig. 1B). Onthe other hand, at 10 �M amitriptyline did not significantlyaffect [3H]NTI saturation binding, whereas at 40 �M it in-creased the KD to 94 5 pM (p � 0.05) without affecting theBmax (1504 55 fmol/mg protein). In functional assays,TCAs induced a concentration-dependent stimulation of[35S]GTP�S binding (Fig. 1C) with different potencies andefficacies. DPDPE, used as a reference agonist, stimulated[35S]GTP�S binding by 97 4% with an EC50 value of 1.3 0.3 nM. Amoxapine behaved as a full agonist displaying amaximal effect equal to that of the selective �-opioid agonistDPDPE and a potency in the low micromolar range (Table 1).The other TCAs tested were 23- to 47-fold less potent thanamoxapine but, with the exception of desipramine, showedonly slightly lower efficacies. Addition of the �-opioid receptorantagonist NTI (10 nM) completely antagonized the stimu-latory effects elicited by the TCAs (Fig. 1D). Each TCA,tested at the same concentration range, failed to significantlystimulate [35S]GTP�S binding in membranes prepared fromnontransfected CHO/K1 cells (result not shown). Moreover,each TCA (1–100 �M) failed to affect [35S]GTP�S binding inmembranes prepared from either CHO/M2 (Bmax � 3200 500 fmol/mg protein) or CHO/M4 (Bmax � 3500 100fmol/mg protein) cells, which were used as control for Gi/o-coupled receptors expressed at similar densities (result notshown).

In intact CHO/DOR cells, amoxapine and amitriptylineinhibited FSK-stimulated cyclic AMP accumulation withEC50 values of 3.4 0.5 and 16.2 0.8 �M, respectively, andmaximal effects corresponding to 41.1 3.2 and 43.5 4.5%decrease of control activity (p � 0.001), respectively (Fig. 2A).These effects were equal to approximately 72% of the maxi-mal inhibitory response elicited by DPDPE (EC50 � 0.7 0.2nM) and were completely antagonized by the addition of 10nM NTI (Fig. 2B).

Besides regulating cyclic AMP formation, �-opioid recep-tors have been shown to modulate other intracellular signal-ing pathways, such as ERK1/2 phosphorylation cascade andthe phosphatidylinositol 3-kinase pathway (Tegeder and Geis-slinger, 2004). These pathways are known to regulate cellproliferation, differentiation, and neuronal survival and havebeen proposed to be molecular targets of antidepressants(Jope and Roh, 2006; Duman et al., 2007). Treatment ofCHO/DOR with amoxapine (10 �M) caused a rapid increaseof the phosphorylation of ERK1/2, Akt, and GSK-3�, whichpeaked at 5 min and remained elevated for at least 30 min(Fig. 3, A and B). Consistent with the stimulation of ERK1/2activity, the drug induced a marked phosphorylation of S6ribosomal protein (pS6rp), a downstream target of ERK1/2,which reached a plateau at approximately 15 min and lastedfor at least 30 min. The stimulatory effects on ERK1/2 andGSK-3� phosphorylations were concentration-dependentwith EC50 values of 5.1 0.4 and 4.4 0.6 �M, respectively,

Fig. 1. Interaction of TCAs with �-opioid receptors. A, displacement ofspecific [3H]NTI binding in CHO/DOR membranes by TCAs. The con-centration of the radioligand was 0.2 nM. Values are the mean S.E.M. of three experiments for each drug. B, Scatchard plot of[3H]NTI saturation binding carried out in the absence (control) and inthe presence of either 10 �M amoxapine or amitriptyline at 10 and 40�M. Values are the mean of three experiments. C, concentration-dependent stimulation of [35S]GTP�S binding by DPDPE and differentTCAs. Values are the mean S.E.M. of three or four experiments foreach drug. D, blockade by NTI of TCA-induced stimulation of [35S]GTP�Sbinding. Cell membranes were preincubated with either vehicle or 10 nMNTI and with either vehicle, amoxapine (final concentration, 10 �M), orthe other TCAs (each at the final concentration of 100 �M) for 15 minat 30°C. Thereafter, the reaction was started by the addition of 1.0 nM

[35S]GTP�S. Values are expressed as percentage of control (vehicle vehi-cle) and are the mean S.E.M. of three experiments. ���, p � 0.001; ns, notsignificantly different versus control by ANOVA followed by Dunnett’s test.

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and completely antagonized by 10 nM NTI (Fig. 3, C–J).Moreover, amoxapine (10 �M) failed to affect ERK1/2 andGSK-3� phosphorylations in nontransfected CHO-K1 cells(result not shown). The maximal stimulations of ERK1/2 andGSK-3� phosphorylations corresponded to 8.0- and 2.4-foldincrease of control values (p � 0.001), respectively, and wereequal to 66 and 70% of the maximal stimulations elicited byDPDPE, respectively. At the concentrations tested, amoxap-ine failed to affect the levels of total ERK1/2 and GSK-3�proteins (Fig. 3, E, G, and I).

Like amoxapine, amitriptyline caused a concentration-de-pendent stimulation of ERK1/2 and GSK-3� phosphorylationwith EC50 values of 10.7 0.9 and 9.0 0.5 �M, respec-tively, and Emax values corresponding to 550 70 and 90

10% increase of control values (p � 0.001), respectively (Fig.4). The addition of NTI (10 nM) significantly attenuated thestimulatory effect of amitriptyline on ERK1/2 phosphoryla-tion and completely blocked the induction of GSK-3� phos-phorylation elicited by the antidepressant (Fig. 4).

Effects of Amoxapine in Rat and Human Brain. Inmembranes of rat dorsal striatum and nucleus accumbens,amoxapine maximally stimulated [35S]GTP�S binding by35 3% (p � 0.01) and 28 5% (p � 0.05) increase of control,respectively (Emax values were equal to 51 and 39% of theDPDPE Emax, respectively), with EC50 values of 5.5 1.3and 3.5 1.0 �M, respectively (Fig. 5A). The use of receptorsubtype-selective antagonists indicated that the stimulatoryeffect of amoxapine (30 �M) was mediated by �-opioid recep-tors. In fact, it was antagonized by NTI with a Ki value of25 5 pM, which is similar to the affinity of the antagonistfor �-opioid receptors. In contrast, the selective �-opioid re-ceptor antagonist nor-BNI was severalfold less potent thanNTI, displaying a Ki of 3.2 0.8 nM (Fig. 5B). This value isapproximately 100-fold higher than nor-BNI affinity for the�-opioid receptor and close to its affinity for �-opioid receptors(Metcalf and Coop, 2005). The selective �-opioid receptorantagonist CTAP failed to cause a significant inhibition up to1 �M (Fig. 5B), a concentration that was sufficient to com-pletely block the stimulatory effect elicited by the �-opioidreceptor agonist DAMGO (100 nM) (result not shown). Noneof the antagonists significantly affected [35S]GTP�S bindingper se. In rat nucleus accumbens, amoxapine inhibited FSK-stimulated adenylyl cyclase activity (18 0.9% reduction ofcontrol activity, p � 0.05, equal to 68% of the DPDPE Emax)with an EC50 of 4.5 0.7 �M (Fig. 5C). This effect wascompletely blocked by the addition of 3 nM NTI. Finally, inmembranes of postmortem human frontal cortex amoxapinestimulated [35S]GTP�S binding by 32 6% (p � 0.05; equalto 48% of DPDPE Emax) with an EC50 value of 3.6 0.8 �M(Fig. 5D). This response also was blocked by the addition of 3nM NTI (Fig. 5E).

Effects of TCAs in CHO/KOR. In receptor binding as-says, TCAs completely displaced specific [3H]diprenorphinebinding with a rank order of potencies opposite to that ob-served at CHO/DOR. In fact, amitriptyline, nortriptyline,desipramine, and imipramine showed Ki values in the lowmicromolar range (1.8–3.3 �M), whereas amoxapine wasmuch less potent (Fig. 6A; Table 2). Scatchard analysis of[3H]diprenorphine saturation binding data indicated thatamitriptyline (10 �M) markedly decreased the affinity of theradioligand (KD value increased from 101 9 to 340 20pM; p � 0.05), whereas amoxapine (10 �M) caused only aslight reduction of this parameter (KD 128 16 pM) (Fig.

Fig. 2. Amoxapine and amitriptyline inhibit cyclic AMP accumulation inCHO/DOR. A, cells prelabeled with [3H]adenine were incubated withFSK (10 �M) and the indicated concentrations of DPDPE, amoxapine,and amitriptyline for 15 min. Values are expressed as percentage ofcontrol and are the mean S.E.M. of three experiments. B, cells werepreincubated for 5 min with and without NTI (10 nM) and then exposedto FSK (10 �M) with vehicle, amoxapine (30 �M), or amitriptyline (50�M) for 15 min. Values are expressed as percentage of control and are themean S.E.M. of three experiments. ���, p � 0.001; ns, not significantlydifferent versus control by ANOVA followed by Dunnett’s test.

TABLE 1Potencies and efficacies of TCAs in stimulating �35S�GTP�S binding in CHO/DOR cells

�35S�GTP�S Assay Cyclic AMP Assay

Kia EC50 Emax %Emax

b EC50 Emax %Emaxb

�M �M �M

Amoxapine 2.5 0.5 0.98 0.08 87 2 96 3 3.4 0.5 42 3 72 2Amitriptyline 28.1 1.2 23.5 1.2 72 3 79 5 16.2 0.8 43 4 74 4Nortriptyline 30.0 1.0 22.6 2.0 77 2 85 3Desipramine 40.1 2.5 35.5 3.5 53 2 59 3Imipramine 49.8 3.5 46.8 4.0 69 3 76 4

a Determined by radioligand binding displacement.b Percentage of maximal stimulation with respect to that elicited by DPDPE. Values are the mean S.E.M. of at least three experiments.

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6B). Both drugs failed to change the Bmax of [3H]diprenor-phine (values were control 1470 35, amitriptyline 1530 45, and amoxapine 1440 30 fmol/mg protein).

In CHO/KOR membranes, TCAs elicited a concentration-dependent stimulation of [35S]GTP�S binding with a rankorder of potency consistent with their receptor affinity. Thus,amitriptyline, nortriptyline, desipramine, and imipramineshowed EC50 values between 1.5 and 4.8 �M, whereas theamoxapine concentration-response curve did not reach a pla-teau at concentrations as high as 300 �M (Fig. 6C). The Emax

values of TCAs ranged from 76 to 85% of that displayed bythe full �-opioid agonist (�)-U50,488 (Emax � 183 8%increase of control value; EC50 � 0.60 0.08 nM) (Fig. 6C;Table 2). In CHO/KOR cells, amitriptyline, nortriptyline,desipramine, and imipramine inhibited FSK-stimulated cy-clic AMP accumulation by approximately 50% with EC50

values close to those observed in [35S]GTP�S binding assays(Fig. 6E; Table 2). The TCA Emax values corresponded to 70to 78% of that of (�)-U50,488, which inhibited cyclic AMPaccumulation by 72 6% with an EC50 of 0.31 0.05 nM. Incontrast, amoxapine displayed a weaker agonist activity andmaximally inhibited cyclic AMP accumulation by 30% at 100�M (Fig. 6E). Both functional responses elicited by TCAswere completely blocked by nor-BNI (100 nM), which had noeffect per se (Fig. 6, D and F).

In CHO/KOR cells, amitriptyline (1–50 �M) induced aconcentration-dependent stimulation of ERK1/2 phosphor-ylation with an EC50 value of 4.5 0.8 �M and an Emax

corresponding to 570 95% increase of control value (p �0.001) (Fig. 7, A and B). The addition of nor-BNI (100 nM)

Fig. 3. Amoxapine stimulates ERK1/2 and phosphatidylinositol 3-kinasesignaling pathways in CHO/DOR cells. A, time course of amoxapine-induced phosphorylation of ERK1/2 (pERK1/2), pS6 ribosomal protein(pS6rp), Akt (pAkt), and GSK-3� (pGSK). Cells were incubated withamoxapine (10 �M) for the indicated time periods, and phosphorylatedproteins were measured in cell extracts by Western blot. B, densitometricanalysis of pERK1/2, pS6rp, pAkt, and pGSK bands normalized to thedensity of actin. Values are reported as percentage of the correspondingvehicle-treated sample (zero time). Values are the mean S.E.M. of threeexperiments. C, concentration-dependent stimulation of ERK1/2 phos-phorylation by amoxapine. Cells were incubated in the presence of theindicated concentrations of amoxapine for 5 min, and pERK1/2 and totalERK1/2 were measured in the cell extracts. D, densitometric analysis ofpERK1/2 normalized to the density of total ERK1/2. Values are reportedas percentage of control and are the mean S.E.M. of three experiments.E, antagonism of amoxapine-induced ERK1/2 phosphorylation by NTI.Cells were preincubated for 5 min with either vehicle or 10 nM NTI andthen exposed to either vehicle or 10 �M amoxapine for additional 5 min.F, densitometric analysis of pERK1/2 normalized to the density of totalERK1/2. Values are reported as percentage of control and are the mean S.E.M. of three experiments. G, concentration-dependent stimulation ofGSK-3� phosphorylation by amoxapine. Cells were incubated in thepresence of the indicated concentrations of amoxapine for 5 min, andpGSK and total GSK-3� (GSK) were measured in the cell extracts.H, densitometric analysis of pGSK normalized to the density of totalGSK. Values are reported as percentage of control and are the mean S.E.M. of three experiments. I, antagonism of amoxapine-inducedGSK-3� phosphorylation by NTI. Cells were preincubated for 5 min with

either vehicle or 10 nM NTI and then exposed to either vehicle or 10 �Mamoxapine for additional 5 min. J, densitometric analysis of pGSK nor-malized to the density of total GSK. Values are reported as percentage ofcontrol and are the mean S.E.M. of three experiments. ���, p � 0.001;��, p � 0.01; �, p � 0.05; ns, not significantly different versus control byANOVA followed by Dunnett’s test.

Fig. 4. Amitriptyline induces a concentration-dependent increase of phos-pho-ERK1/2 and phospho-GSK-3� in CHO/DOR. Cells were preincubatedfor 5 min with either vehicle or 10 nM NTI and then exposed to theindicated concentrations of amitriptyline for additional 10 min. Cell ex-tracts were then subjected to Western blot for the analysis of eitherpERK1/2 and total ERK1/2 (A) or pGSK and total GSK (C). Diagrams inB and D show the densitometric analysis of pERK1/2 normalized to thedensity of total ERK1/2 and of pGSK normalized to the density of GSK,respectively. Values are reported as percentage of control and are themean S.E.M. of four experiments. ���, p � 0.001; ��, p � 0.01; �, p �0.05 versus control (vehicle vehicle) by ANOVA followed by Dunnett’stest.

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effectively antagonized the stimulatory effect elicited byamitriptyline.

Effects of TCAs in C6 Glioma Cells. C6 glioma cells areknown to endogenously express �-opioid receptors coupled tostimulation of ERK1/2 phosphorylation (Bohn et al., 2000).Moreover, it has been recently reported that in this cell lineTCAs induced ERK1/2 activation through a monoamine-in-

Fig. 5. �-Opioid agonist activity of amoxapine in brain membranes.A, stimulation of [35S]GTP�S binding in rat dorsal striatum and nu-cleus accumbens. Values are expressed as percentage of control andare the mean S.E.M. of four experiments performed in three differ-ent tissue preparations. B, antagonism of amoxapine (30 �M)-stimulated[35S]GTP�S binding by NTI, nor-BNI, and CTAP in rat dorsal striatum.Values are reported as percentage of the stimulation elicited by amoxap-ine and are the mean S.E.M. of three experiments. C, effects of amox-apine on FSK-stimulated cyclic AMP formation in rat nucleus accumbensin the absence (vehicle) and in the presence of 3 nM NTI. Values areexpressed as percentage of control and are the mean S.E.M. of threeexperiments. D, stimulation of [35S]GTP�S binding in membranes ofpostmortem human frontal cortex. Values are expressed as percentage ofcontrol and are the mean S.E.M. of three experiments performed ontwo distinct tissue preparations. E, antagonism of amoxapine (Amox) (30�M) stimulation of [35S]GTP�S binding by NTI (3 nM) in membranes ofhuman frontal cortex. Values are expressed as percentage of control andare the mean S.E.M. of three experiments performed on three distincttissue preparations. �, p � 0.05; ns, not significantly different versuscontrol by ANOVA followed by Dunnett’s test.

Fig. 6. Interaction of TCAs with �-opioid receptors. A, displacement ofspecific [3H]diprenorphine binding in CHO/KOR membranes by TCAs.The concentration of the radioligand was 0.2 nM. Values are the mean S.E.M. of three experiments for each drug. B, Scatchard plot of [3H]di-prenorphine saturation binding carried out in the absence (control) and inthe presence of either 10 �M amitriptyline or 10 �M amoxapine. Valuesare the mean of three experiments. C, concentration-dependent stimula-tion of [35S]GTP�S binding by (�)-U50,488, amoxapine, amitriptyline,nortriptyline, desipramine, and imipramine in CHO/KOR cell mem-branes. Values are the mean S.E.M. of three experiments for each drug.D, blockade of TCA-induced stimulation of [35S]GTP�S binding by nor-BNI. Membranes were preincubated with either vehicle or 100 nM nor-BNI in the absence and in the presence of amoxapine (100 �M) or theother TCAs (each at the final concentration of 20 �M) for 15 min at 30°Cbefore starting the reaction by adding [35S]GTP�S. Values are expressedas percentage of control (vehicle vehicle) and are the mean S.E.M. ofthree experiments. E, inhibition of FSK-stimulated cyclic AMP accumu-lation by TCAs. Cells prelabeled with [3H]adenine were incubated at 37°Cfor 15 min with FSK (10 �M) in the presence of the indicated concentra-tions of either (�)-U50,488 or TCAs. Values are expressed as percentageof control and are the mean S.E.M. of three experiments. F, antagonismof (�)-U50,488- and TCA-induced inhibition of FSK-stimulated cyclicAMP accumulation by nor-BNI. Cells were preincubated for 5 min witheither vehicle or 100 nM nor-BNI and then treated with FSK (10 �M) inthe presence of either (�)-U50,488 (100 nM), amoxapine (100 �M), orother TCAs (each at 20 �M). Values are expressed as percentage ofcontrol and are the mean S.E.M. of three experiments. ���, p � 0.001;��, p � 0.01; ns, not significantly different versus control by ANOVAfollowed by Dunnett’s test.

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dependent mechanism (Hisaoka et al., 2007). Therefore, itwas of interest to investigate whether �-opioid receptorscould mediate the actions of TCAs on ERK1/2 signaling inthis cell system. As shown in Fig. 7, C and D, a brief exposureof C6 cells to amitriptyline (15 �M) caused a stimulation of

ERK1/2 phosphorylation, which corresponded to a 5.7-foldincrease of control value (p � 0.001). The addition of nor-BNI(100 nM) reduced the amitriptyline stimulatory effect by 70%(p � 0.001). When ERK1/2 activity was measured after im-munoprecipitation and determination of Elk-1phosphoryla-tion, it was found that amitriptyline (15 �M) stimulated thekinase activity by 4.8-fold (p � 0.001) (Fig. 7, E and F) andthat this response was reduced by approximately 50% afterpretreatment with 100 nM nor-BNI (p � 0.01).

Effects of Amitriptyline in Rat Nucleus Accumbens.In membranes of rat nucleus accumbens, amitriptylinecaused a slight but significant inhibition of FSK-stimulatedcyclic AMP formation with an EC50 value of 6.2 0.8 �M andan Emax corresponding to 12 3% decrease of control activity(p � 0.05) (Fig. 8A). The inhibitory effect was completelyantagonized by 10 nM nor-BNI (result not shown). Whencombined with (�)-U50,488 (300 nM), amitriptyline caused aconcentration-dependent reduction of the inhibitory effectelicited by the full �-opioid agonist, bringing the enzymeactivity to the level determined by its maximal effect (Fig.8A). In the same membrane preparations, (�)-U50,488 max-imally inhibited FSK-stimulated cyclic AMP formation by25 4% with an EC50 of 57 3 nM. In the presence of 30 �Mamitriptyline, the (�)-U50,488 concentration-response curvewas shifted to the right by 7.9-fold, yielding a Ki value of4.3 0.6 �M (Fig. 8B).

Effects of TCAs in CHO/MOR. All the TCAs tested com-pletely displaced specific [3H]diprenorphine bound to �-opi-oid receptors with relatively low potencies (Fig. 9A). Theestimated Ki values were amitriptyline, 30 2 �M; nortrip-tyline, 22 3 �M; desipramine, 23 4 �M; imipramine, 25 3 �M; and amoxapine, 44 5 �M. Scatchard analysis of[3H]diprenorphine saturation binding data indicated that, atthe concentration of 30 �M, both amitriptyline and amoxap-ine decreased the affinity of the radioligand (KD values werecontrol, 118 10 pM; amitriptyline, 370 18 pM, p � 0.05;amoxapine, 275 15 pM, p � 0.05) without significantlychanging the Bmax (control, 600 30 fmol/mg protein; ami-triptyline, 620 35 fmol/mg protein; amoxapine, 585 29fmol/mg protein) (Fig. 9B). In [35S]GTP�S binding assays, theTCAs caused a small and not significant stimulatory effect,which occurred at concentrations greater than 10 �M (Fig.9C). Under the same conditions, the selective �-opioid recep-tor agonist DAMGO increased [35S]GTP�S binding by 180 12% (p � 0.001) with an EC50 value of 29 5 nM. Whenexamined in the presence of 30 nM DAMGO, the TCAs dis-played a weak antagonistic activity with the following Ki

values: amitriptyline, 36 4 �M; nortriptyline, 25 3 �M;

TABLE 2Potencies and efficacies of TCAs in CHO/KOR cells

�35S�GTP�S Assay Cyclic AMP Assay

Kia EC50 Emax %Emax

b EC50 Emax %Emaxb

�M �M �M

Amitriptyline 1.8 0.6 1.5 0.2 140 4 76 3 5.7 0.5 55 4 78 3Nortriptyline 3.2 0.9 2.4 0.5 146 3 77 5 7.4 0.4 51 5 73 4Desipramine 3.3 0.4 4.8 0.4 169 5 85 4 6.7 0.8 53 3 77 2Imipramine 2.5 0.6 2.3 0.5 160 6 84 6.0 9.9 1.2 49 6 70 3Amoxapine 32.5 2.7 �100 �50

a Determined by radioligand binding displacement.b Percentage of the maximal effect with respect to that elicited by (�)-U50,488. Values are the mean S.E.M. of at least three experiments.

Fig. 7. Amitriptyline enhances ERK1/2 activity in CHO/KOR and C6glioma cells. A, concentration-dependent stimulation of ERK1/2 phos-phorylation in CHO/KOR. Cells were incubated for 5 min with eithervehicle or 100 nM nor-BNI and then exposed to the indicated concentra-tions of amitriptyline for 10 min. B, densitometric analysis of pERK1/2normalized to the density of total ERK1/2. Values are expressed aspercentage of control (vehicle vehicle) and are the mean S.E.M. offour experiments. ���, p � 0.001; ��, p � 0.01; �, p � 0.05 versus control.C, induction of ERK1/2 phosphorylation by amitriptyline in C6 gliomacells. Cells were incubated for 5 min with either vehicle or 100 nMnor-BNI and then treated with either vehicle or 15 �M amitriptyline(Amitrip) for 10 min. D, densitometric analysis of pERK1/2 normalized tothe density of total ERK1/2. Values are expressed as percentage of control(vehicle vehicle) and are the mean S.E.M. of three experiments.E, enhancement of ERK1/2 activity by amitriptyline in C6 glioma cells.Cells were treated as described in C, and pERK1/2 activity was assayedafter immunoprecipitation by measuring exogenous Elk-1 phosphoryla-tion (pElk-1). F, densitometric analysis of pElk-1 normalized to the den-sity of ERK1/2. Values are expressed as percentage of control (vehicle vehicle) and are the mean S.E.M. of three experiments. ���, p � 0.001;�, p � 0.05 versus control by ANOVA followed by Newman-Keuls test.

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desipramine, 27 4 �M; imipramine, 26 5 �M; and amox-apine, 74 7 �M (Fig. 9D).

DiscussionIn the present study, we show for the first time that widely

used TCAs, such as amoxapine, amitriptyline, nortriptyline,desipramine, and imipramine, exert direct agonist activity atdistinct opioid receptor subtypes. In CHO cells individuallyexpressing the human receptors these drugs display differ-ential affinity for �-, �-, and �-opioid receptors. Thus, amox-apine, an antidepressant with rapid onset of therapeuticefficacy and atypical antipsychotic properties, preferentiallybound to the �-opioid receptor, whereas amitriptyline, nor-triptyline, imipramine, and desipramine showed higher se-lectivity for the �-opioid receptor. All the TCAs examined hadrelatively low affinity for the �-opioid receptor. At each opioidreceptor subtype, the TCAs completely displaced the specificbinding of the radioligands and decreased the affinities of theradioligands without significantly affecting the Bmax values.These data indicated that TCAs interacted with the opioidreceptors as competitive ligands.

In agreement with the radioligand binding data, TCAsshowed differential agonist activity at the distinct opioidreceptor subtypes. In fact, the drugs stimulated �- and �-opi-oid receptors without significant agonist effects at �-opioidreceptor. Moreover, important differences were observed in

Fig. 9. Interaction of TCAs with �-opioid receptors. A, displacement ofspecific [3H]diprenorphine binding in CHO/MOR-1 cell membranes byTCAs. The concentration of the radioligand was 0.2 nM. Values are themean S.E.M. of three experiments for each drug. B, Scatchard plot of[3H]diprenorphine saturation binding carried out in the absence (control)and in the presence of either 30 �M amitriptyline or 30 �M amoxapine.Values are the mean of three experiments. C, effects of TCAs on [35S]GTP�Sbinding in CHO/MOR-1 cell membranes. The stimulatory effect of the full�-opioid agonist DAMGO is also reported. Values are the mean S.E.M. ofthree experiments for each drug. D, effects of TCAs on DAMGO-stimulated[35S]GTP�S binding in CHO/MOR-1 cell membranes. Membranes were pre-incubated with the indicated concentrations of the TCAs in the absence andin the presence of 30 nM DAMGO and then assayed for [35S]GTP�S binding.Values are expressed as percentage of the net DAMGO stimulatory effect ateach drug concentration and are the mean S.E.M. of three experiments.

Fig. 8. Amitriptyline inhibits FSK-stimulated adenylyl cyclase activityand antagonizes (�)-U50,488 inhibitory effect in rat nucleus accumbens.A, the enzyme activity stimulated by 10 �M FSK was assayed at theindicated concentrations of amitriptyline in the presence of either vehicleor 300 nM (�)-U50,488. Values are expressed as percentage of controlvalue (vehicle vehicle) and are the mean S.E.M. of four experiments.B, antagonism of (�)-U50,488 induced inhibition of FSK-stimulated ad-enylyl cyclase activity by amitriptyline. The enzyme activity was assayedat the indicated concentrations of (�)-U50,488 in the presence of eithervehicle or 30 �M amitriptyline. Values are expressed as percentage ofcontrol activity (vehicle vehicle) and are the mean S.E.M. of threeexperiments.

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the drug potencies and efficacies at �- and �-opioid receptors.Thus, amoxapine was the most potent and efficacious �-opi-oid receptor agonist but a weak stimulant of �-opioid recep-tors. In contrast, classic TCAs displayed higher potencies at�- than �-opioid receptors. In line with the present study,Isenberg and Cicero (1984) reported that in rat brain mem-branes amoxapine inhibited the binding of the �-opioid re-ceptor agonist [3H][D-Ala2,D-Leu5]enkephalin 5-fold more po-tently than that of the nonselective opioid receptor ligand[3H]naltrexone. The preferential activity of classic TCAs at�-opioid receptors correlates with previous radioligand bind-ing studies reporting a higher selectivity of these drugs for�- over �- and �-opioid binding sites in the human thalamicarea (Wahlstrom et al., 1994).

The direct agonist activity of amoxapine was characterizedat both cloned and native �-opioid receptors. In CHO/DORcells, the drug mimicked the responses to DPDPE in differentfunctional assays, including stimulation of [35S]GTP�S bind-ing, inhibition of cyclic AMP accumulation, activation of theERK1/2 pathway, and induction of Akt and GSK-3� phospho-rylations. In addition, amoxapine was capable of activating�-opioid receptors endogenously present in rat dorsal stria-tum and nucleus accumbens and in human frontal cortex,indicating that the agonist activity was not limited to heter-ologous cell expression systems. Although in CHO/DOR cellsamoxapine stimulated [35S]GTP�S binding almost as effica-ciously as DPDPE, in all the other functional assays it con-sistently showed Emax values lower than those of the fullagonist, indicating a partial agonist activity at the �-opioidreceptor. Likewise, the maximal responses elicited by classicTCAs at either �- or �-opioid receptors were significantlylower than those of the corresponding full agonist, suggestingthat these drugs also act as partial agonists.

Because TCAs are known to interact with different neuro-transmitter receptor systems and to affect several cellularevents, it was important to assess the specificity of theiragonist activity. In CHO/DOR cells and rat and human brain,the amoxapine responses were completely blocked by lownanomolar concentrations of NTI, indicating the involvementof �-opioid receptors. With regard to the classic TCAs, inCHO/DOR cells the stimulation of [35S]GTP�S binding wastotally blocked by NTI, and in CHO/KOR cells both stimula-tion of [35S]GTP�S binding and inhibition of cyclic AMPaccumulation were completely antagonized by nor-BNI, indi-cating that the effects were specifically mediated by either�- or �-opioid receptors, respectively. The stimulations ofERK1/2 phosphorylation by amitriptyline observed in CHO/DOR, CHO/KOR, and C6 glioma cells were largely reducedby the opioid receptor antagonists but not completely antag-onized, particularly at concentrations of the antidepressanthigher than 10 to 15 �M. This indicates that amitriptylinecould also induce ERK1/2 phosphorylation through an opioidreceptor-independent mechanism. The molecular events me-diating the latter effect are still unknown and require furtherinvestigation.

TCAs are known to accumulate in the brain where theyreach concentrations severalfold higher than those in theblood. The plasma concentrations of the TCAs tested rangefrom 0.2 to 1.6 �M (Baldessarini, 2006), whereas their brain-to-plasma ratios have been reported to be 10 to 35:1(Glotzbach and Preskorn, 1982). In rats, the administrationof 10 to 15 mg/kg desipramine or 20 mg/kg amoxapine pro-

duced brain concentrations equal to 20 to 40 �M and 13 �M,respectively (Biegon and Samuel, 1980; Kobayashi et al.,1992). Thus, the observed potencies of TCAs as �- and �-opi-oid receptor agonists are consistent with the brain concen-trations reached by these drugs at clinically relevant doses.

The present demonstration that TCAs directly stimulate�- and �-opioid receptors raises the possibility that this prop-erty may contribute to their analgesic activity. Acute andchronic administrations of TCAs have been shown to displayantinociceptive properties in various acute experimentalpain models both in animals and humans, and in several ofthese tests a sensitivity to blockade by naloxone has beenobserved (Mico et al., 2006). Recent studies have highlightedthe participation of distinct opioid receptor subtypes. Thus,in the acetic acid-induced abdominal constriction test, theanalgesic effect of amitriptyline was more potently antago-nized by NTI than naloxone, indicating the preferential in-volvement of �-opioid receptors (Gray et al., 1998). The anti-allodynic properties of chronic nortriptyline treatment wasabsent in mice deficient of �-opioid receptors (Benbouzid etal., 2008b), and both �- and �-opioid receptor antagonistsblocked the attenuation of neuropathic allodynia elicited bychronic TCA treatment (Benbouzid et al., 2008a). Moreover,supraspinal �- and spinal �-opioid receptors have been pro-posed to be involved in the antihyperalgesic effect of chroni-cally administered clomipramine in a mononeuropathic painmodel in rats (Marchand et al., 2003). Although it is possiblethat these effects may result from indirect actions of TCAs onthe opioidergic system (i.e., enhanced release of endogenousopioid peptides), the present data support the idea that atleast a portion of the TCA analgesic effect may involve adirect agonist activity at �- and �-opioid receptors. This directmechanism may operate in conjunction with the primaryaction of these drugs on noradrenergic and serotoninergicsystems (Mico et al., 2006).

It is also possible that the opioid agonist activity of TCAs,particularly at �-opioid receptors, may contribute to theirmood-elevating effects. Genetic and pharmacological studieshave shown that �-opioid receptors exert antidepressant ac-tivity (Filliol et al., 2000; Jutkiewicz, 2006). Cellular studieshave reported that activation of �-opioid receptors inducesmitogenic responses (Wilson et al., 1997) and promotes neu-roprotection and neurogenesis (Narita et al., 2006), whichseem to be critical for the action of antidepressants (Dumanet al., 2000). At the molecular level, these neurotrophic ef-fects occur through recruitment of downstream intracellularmolecules and phosphorylation events, particularly those as-sociated with ERK1/2 and Akt/GSK-3� signaling pathways(Tegeder and Geisslinger, 2004), which, as shown in thepresent study, can be regulated by TCAs via activation of�- and �-opioid receptors. Moreover, both ERK1/2 andGSK-3� have been linked to the action of antidepressants(Jope and Roh, 2006; Duman et al., 2007). In C6 glioma cells,amitriptyline and other TCAs have been recently reported toregulate the production of glial cell line-derived neurotrophicfactor, a stimulant of adult neurogenesis, by activatingERK1/2 (Hisaoka et al., 2007). Because the present studyshows that in these cells amitriptyline-induced ERK1/2 stim-ulation is largely mediated by stimulation of �-opioid recep-tors, it is possible that this receptor system also participatesin TCA regulation of the synthesis of glial cell line-derivedneurotrophic factor and other neurotrophic factors.

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The agonist activity at �- and �-opioid receptors may alsobe involved in some TCA adverse effects. For example, theadministration of nonpeptidic �-opioid agonists is known tocause seizure in laboratory animals (Jutkiewicz, 2006),whereas activation of �-opioid receptors may exert anticon-vulsant activity (Loacker et al., 2007). In this context, thehigher potency and efficacy of amoxapine at �- versus �-opi-oid receptors may correlate with the observed greater risk ofseizure associated with amoxapine overdose, compared withother antidepressants (Litovitz and Troutman, 1983). TCAsare also known to induce a dysphoric-agitated state in somepatients (Baldessarini, 2006). In animal models, the admin-istration of �-opioid receptor agonists has been reported tolower mood and exert prodepressive effects, whereas antag-onists produce anxiolytic and antidepressant actions (Magueet al., 2003). Moreover, distinct stress-induced behavioralresponses have been shown to be antagonized by �-opioidreceptor blockers or deletion of the prodynorphin gene, sug-gesting that dynorphin stimulation of �-opioid receptors maymediate stress-induced dysphoria (McLaughlin et al., 2003).Thus, the �-opioid agonist activity may be related to TCA-induced mood worsening and anxiogenic reactions. However,there is also evidence that activation of �-opioid receptorsproduces anxiolytic-like behavior (Wall and Messier, 2000).Moreover, the present study shows that in rat nucleus ac-cumbens amitriptyline behaves as partial agonist at �-opioidreceptors and antagonizes the action of the full agonist (�)-U50,488. Thus, it is possible that in stress-related conditions,where an intense release of dynorphin can overstimulate�-opioid receptors, the partial agonist activity of TCAs mayreduce the level of receptor activation, thus attenuating,rather than enhancing, the aversive emotional consequencesof increased �-opioid transmission.

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Address correspondence to: Pierluigi Onali, Department of Neurosciences,Section of Biochemical Pharmacology, University of Cagliari, Cittadella Uni-versitaria di Monserrato, 09042 Monserrato (CA), Italy. E-mail: [email protected]

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