0022-3565/10/3322-437–445$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 332, No. 2 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 159160/3553965 JPET 332:437–445, 2010 Printed in U.S.A. Amoxapine Inhibits the Delayed Rectifier Outward Kϩ Current in Mouse Cortical Neurons via cAMP/Protein Kinase A Pathways Yan-Lin He, Xiao-Qin Zhan, Guang Yang, Ji Sun, and Yan-Ai Mei Institutes of Brain Science, School of Life Sciences and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, China Received July 30, 2009; accepted November 12, 2009 Downloaded from ABSTRACT Ion channels are known to be modulated by antidepressant pine inhibition of IK. Amoxapine was also found to significantly drugs, but the molecular mechanisms are not known. We have increase intracellular cAMP levels. The effects of amoxapine on shown that the antidepressant drug amoxapine suppresses IK were abolished by preincubation with 5-hydroxytryptamine (5- ϩ rectifier outward K (IK) currents in mouse cortical neurons. At HT) and the antagonists of 5-HT2 receptor. Moreover, intracellular jpet.aspetjournals.org ␮ Ј ␥ a concentration of 10 to 500 M, amoxapine reversibly inhib- application of guanosine 5 -[ thio]-triphosphate increased IK am- ited IK in a dose-dependent manner and modulated both plitudes and prevented amoxapine-induced inhibition. The selec- steady-state activation and inactivation properties. The appli- tive Kv2.1 subunit blocker Jingzhaotoxin-III reduced IK amplitudes cation of forskolin or dibutyryl cAMP mimicked the inhibitory by 30% and also significantly abolished the inhibitory effect of effect of amoxapine on IK and abolished further inhibition by amoxapine. Together these results suggest that amoxapine inhib- amoxapine. N-[2-(p-Bromocinnamylamino)ethyl]-5-iso-quino- its IK in mouse cortical neurons by cAMP/PKA-dependent path- linesulphonamide (H-89), a protein kinase A (PKA) inhibitor, way associated with the 5-HT receptor, and suggest that the ␣ at ASPET Journals on January 16, 2020 augmented IK amplitudes and completely eliminated amoxa- Kv2.1 -subunit may be the target for this inhibition. Tricyclic antidepressants (TCAs), named after their three- kir4.1 channels in astrocytes (Su et al., 2007). Imipramine ring molecular core structure, were the first successful anti- and structurally related compounds can directly inhibit tran- depressants and have been widely used for the treatment of sient KA currents in rat hippocampal neurons, and it was depression and other psychiatric disorders (Chen et al., 2004; hypothesized that there is a specific imipramine binding site Gillman, 2007). The therapeutic effects of these drugs are consisting of an aliphatic and aromatic site on the external generally thought to be mediated by inhibition of specific side of the channel pore (Kuo, 1998). Moreover, imipramine serotonin [5-hydroxytryptamine (5-HT)] and/or norepineph- and amitriptyline can inhibit G protein-activated inwardly rine transporters in the brain (Baldessarini, 2001), whereas rectifying Kϩ channels expressed in Xenopus oocytes (Taka- the adverse side effects of TCAs have been attributed to hashi et al., 2006). In addition, TCAs have been shown to interactions with muscarinic, adrenergic, and histamine re- modulate Ca2ϩ-activated Kϩ channels and ClϪ channels ceptors (Baldessarini, 2001). In addition to these pharmaco- (Terstappen et al., 2001; Maertens et al., 2002). Moreover, logic interactions, TCAs have been reported to exert addi- TCAs are known to alleviate various pain syndromes by tional effects on diverse ion channels. Nortriptyline is reported to overlapping with the local anesthetic site in Naϩ channels be a potent blocker of voltage-gated inwardly rectifying (Song et al., 2008). Although the effects on neurotransmitter transporters are held to underlie the activity of TCAs, it is possible that their affinity for ion channels may modulate the This work was supported in part by the National Basic Research Program therapeutic effects and side effects of these molecules. of China [Grant 2007CB512303]; Shanghai Leading Academic Discipline Project [Grant B111]; and the National Talent Training Fund in Basic Re- Amoxapine is a second-generation TCA previously intro- search of China [Grant J0630643]. duced as an alternative to traditional TCAs because of its Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. shorter onset of action and reduced cardiotoxicity. The mol- doi:10.1124/jpet.109.159160. ecule was found to exert antipsychotic effects in animal mod- ABBREVIATIONS: TCA, tricyclic antidepressant; 5-HT, 5-hydroxytryptamine; IK, delayed rectifier potassium current; PKA, protein kinase A; ANOVA, analysis of variance; PBS, phosphate-buffered saline; HEK, human embryonic kidney; GTP␥S, guanosine 5Ј-[␥-thio]-triphosphate; db-cAMP, dibutyryl cAMP; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide; 4-AP, 4-aminopyridine; C6-ceramide, dihy- droceramide C6; JZTX-III, Jingzhaotoxin-III; DMSO, dimethyl sulfoxide; IA, transient potassium current. 437 438 He et al. els (Greenblatt et al., 1978) and was tested as a single-drug actively proliferating fibroblasts for 48 h. On day 4, cultured cells treatment for psychotic depression. In systematic double- were washed and maintained in fresh medium. All the experiments blind trials, amoxapine was found to be of equal efficacy to were carried out with cortical neurons during 6 to 10 days in culture. that of an antidepressant and an antipsychotic (Anton and Patch-Clamp Recordings. Whole-cell currents of cortex neurons were recorded using a patch-clamp technique. Before I recording, Burch, 1990). A recent study found that the antipsychotic K the culture medium was replaced with a bath solution containing activity of amoxapine was comparable with that of risperi- 140 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1 mM MgCl2, and 0.001 done (Apiquian et al., 2005). Furthermore, because amoxa- mM tetrodotoxin (pH adjusted to 7.4 using NaOH). Soft glass record- pine is now off-patent and can be produced at generic prices, ing pipettes were filled with an internal solution containing 135 mM the drug could potentially rival other typical antipsychotics. potassium gluconate, 10 mM KCl, 10 mM HEPES, 1 mM CaCl2,2mM Therefore, an understanding of the molecular and cellular MgCl2, and 10 mM EGTA (pH adjusted to 7.3 using KOH). The mechanisms underlying the therapeutic effects and/or side pipette resistance is 5 to 7 M⍀ after filling with the internal solution. effects of amoxapine is not of purely academic interest. Al- All the recordings were performed at room temperature (23–25°C). though some reports have suggested that amoxapine may Data Acquisition and Analysis. All the currents were recorded using an Axopatch 200B amplifier (Axon Instruments, Sunnyvale, also target 5-HT or D2 receptors (Nasu et al., 2000), few studies have addressed the molecular mechanism that is CA) operated in voltage-clamp mode. A Pentium (Intel Corporation, responsible for the action of this molecule. Santa Clara, CA) computer connected to the recording equipment with a Digidata 1300 (MDS Analytical Technologies, Sunnyvale, CA) Kϩ channels are extremely diverse in structure and func- analog-to-digital interface. Current was digitally sampled at 100 ␮s tion and have been held to be among the most important (10 kHz). The current signals were filtered by a 3-kHz, three-pole Downloaded from signaling macromolecules both in neuronal and non-neuro- Bessel filter. Currents were corrected on-line for leak and residual ϩ nal cells. In the central nervous system, K channels’ activity capacitance transients by a P/4 protocol. Data acquisition and anal- determines the shape, frequency, and duration of action po- ysis were performed with pClamp 8.01 software (Axon Instruments) tentials (Melishchuk et al., 1998). In addition, Kϩ channels and/or Origin 6.1 (MicroCal, Northampton, MA). Statistical analysis play a major role in setting and maintaining the resting was performed using the Student’s t test with nonpaired comparison or paired comparisons where it was relevant. Values were given as membrane potential, and the modification of this potential jpet.aspetjournals.org ϩ Ϯ Ͻ can alter neuronal excitability by activating Ca2 channels mean S.E.M., with n as the number of cell tested. P value 0.05 and modulating neuronal transmitter release (Roeper and was used to denote the statistical difference between groups. When multiple comparisons were made, data were analyzed by a one-way Pongs, 1996). Indeed, blockade of Kϩ channels has been analysis of variance (ANOVA) test. proposed as a potential therapy for diverse disorders, includ- cAMP Assay. cAMP levels were measured as described previ- ing myasthenia gravis, multiple sclerosis, Huntington’s cho- ously (Clark et al., 2004). In brief, 1 ϫ 105 cells were plated in each ϩ rea, and Alzheimer’s disease. The role of K channels is 35-mm dish and grown to confluence. Cells were washed with 1 ml of underscored by the accumulation of potassium ion that takes medium and preincubated at 37°C for 10 min in the presence of the at ASPET Journals on January 16, 2020 place during repetitive neuronal firing, and anticonvulsant phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (2.5 mM) drugs modulating Kϩ channel activity have potential as a (Sigma-Aldrich, St. Louis, MO). Cells were incubated for an addi- means to control seizure activity (Liu et al., 2007). Although tional 3 min at 37°C with or without amoxapine-varying concentra- ϩ ␮ some TCAs have previously been reported to target K chan- tions of 10, 50, and 100 M. The medium was removed, and 0.5 ml of nels, few investigations have addressed the possibility that HCl (0.1 M) with 0.8% Triton X-100 (Sigma-Aldrich) was added to ϩ the plates. After 10-min incubation at room temperature, the lysate amoxapine might modify the activity of voltage-gated K was removed from the plates and centrifuged for 2 min.