Abstract
Atomoxetine and reboxetine are commonly used as selective norepinephrine reuptake inhibitors (NRIs) for the treatment of attention-deficit/hyperactivity disorder and depression, respectively. Furthermore, recent studies have suggested that NRIs may be useful for the treatment of several other psychiatric disorders. However, the molecular mechanisms underlying the various effects of NRIs have not yet been sufficiently clarified. G-protein-activated inwardly rectifying K+ (GIRK or Kir3) channels have an important function in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to be a potential treatment for several neuropsychiatric disorders and cardiac arrhythmias. In this study, we investigated the effects of atomoxetine and reboxetine on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2, GIRK2, or GIRK1/GIRK4 subunits, extracellular application of atomoxetine or reboxetine reversibly reduced GIRK currents. The inhibitory effects were concentration-dependent, but voltage-independent, and time-independent during each voltage pulse. However, Kir1.1 and Kir2.1 channels were insensitive to atomoxetine and reboxetine. Atomoxetine and reboxetine also inhibited GIRK currents induced by activation of cloned A1 adenosine receptors or by intracellularly applied GTPĪ³S, a nonhydrolyzable GTP analogue. Furthermore, the GIRK currents induced by ethanol were concentration-dependently inhibited by extracellularly applied atomoxetine but not by intracellularly applied atomoxetine. The present results suggest that atomoxetine and reboxetine inhibit brain- and cardiac-type GIRK channels, revealing a novel characteristic of clinically used NRIs. GIRK channel inhibition may contribute to some of the therapeutic effects of NRIs and adverse side effects related to nervous system and heart function.
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INTRODUCTION
Atomoxetine (originally named tomoxetine) and reboxetine are commonly used as selective norepinephrine reuptake inhibitors (NRIs) for the treatment of attention-deficit/hyperactivity disorder and depression, respectively (HajĆ³s et al, 2004; Garland and Kirkpatrick 2004; Simpson and Plosker, 2004; Supplementary Figure S1). Their clinical efficacy is hypothesized to be linked mainly with potent inhibition of presynaptic norepinephrine transporters (Wong et al, 2000; HajĆ³s et al, 2004; Simpson and Plosker, 2004). Furthermore, recent studies have suggested that the drugs are potentially useful for the treatment of several other psychiatric conditions, including anxiety disorders, eating disorders, substance use disorders, and narcolepsy (Kadhe et al, 2003; HajĆ³s et al, 2004; Szerman et al, 2005; McElroy et al, 2007; Geller et al, 2007; Wilens et al, 2008). However, the molecular mechanisms underlying the various effects of NRIs have not yet been sufficiently clarified.
G-protein-activated inwardly rectifying K+ (GIRK) channels(also known as Kir3 channels) are members of a major subfamily of inwardly rectifying K+ (Kir) channels that include seven subfamilies (Reimann and Ashcroft, 1999). Four GIRK channel subunits have been identified in mammals (Kubo et al, 1993b; Krapivinsky et al, 1995; Lesage et al, 1995). Neuronal GIRK channels are predominantly heteromultimers composed of GIRK1 and GIRK2 subunits in most brain regions or homomultimers composed of GIRK2 subunits in the substantia nigra (Lesage et al, 1995; Karschin et al, 1996; Liao et al, 1996; Inanobe et al, 1999), whereas atrial GIRK channels are heteromultimers composed of GIRK1 and GIRK4 subunits (Krapivinsky et al, 1995). The channels are activated by various Gi-protein-coupled receptors, such as M2 muscarinic, Ī±2 adrenergic, D2 dopaminergic, opioid, nociceptin/orphanin FQ, CB1 cannabinoid, and A1 adenosine receptors, through the direct action of G-protein Ī²Ī³ subunits (North, 1989; Dascal, 1997; Kobayashi and Ikeda, 2006). Additionally, ethanol activates GIRK channels independently of G-protein-coupled signaling pathways (Kobayashi et al, 1999; Lewohl et al, 1999). GIRK channels have an important function in regulating neuronal excitability, synaptic transmission, and heart rate (North, 1989; LĆ¼scher et al, 1997; Signorini et al, 1997; Kuzhikandathil and Oxford, 2002; Kovoor et al, 2001). Furthermore, recent studies have suggested that GIRK channel modulation has the potential for treating several neuropsychiatric disorders and cardiac arrhythmias (Hashimoto et al, 2006; Kobayashi and Ikeda 2006; Cruz et al, 2008). Therefore, GIRK channel modulators may affect various brain and cardiac functions. In this study, the effects of atomoxetine and reboxetine on GIRK channels were examined using the Xenopus oocyte expression assay.
MATERIALS AND METHODS
Preparation of Specific mRNAs
Plasmids containing the entire coding sequences for the mouse GIRK1, GIRK2, and GIRK4 channel subunits and the Xenopus A1 adenosine receptor (A1R) were obtained previously (Kobayashi et al, 1995, 1999, 2000, 2002). cDNAs for rat Kir1.1 in pSPORT (Ho et al, 1993) and mouse Kir2.1 in pcDNA1 (Kubo et al, 1993a) were generously provided by Dr Steven C Hebert (Yale University) and Dr Lily Y Jan (University of California, San Francisco), respectively. These plasmids were linearized by digestion with the appropriate enzymes as described previously (Ho et al, 1993; Kubo et al, 1993a; Kobayashi et al, 2000). The specific mRNAs were synthesized in vitro using the mMESSAGE mMACHINE in vitro transcription kit (Ambion, Austin, TX, USA).
Electrophysiological Analysis
Adult female Xenopus laevis frogs (Copacetic, Soma, Aomori, Japan) were anesthetized by immersion in water containing 0.15% tricaine (Sigma-Aldrich, St Louis, MO, USA). A small incision was made on the abdomen to remove several ovarian lobes from the frogs, which were humanely killed after the final collection. All procedures for the care and treatment of animals were carried out in accordance with National Institutes of Health guidelines and were approved by our Institutional Animal Care and Use Committee. Xenopus oocytes (Stages V and VI) were manually isolated from the ovary and maintained in Barth's solution (Kobayashi et al, 2002). Oocytes were injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 combinations (each 0.15āng), GIRK2 (1āng), Kir1.1 (2āng), Kir2.1 (0.3āng), or A1R (5āng). The oocytes were incubated at 19Ā°C in Barth's solution and manually defolliculated after treatment with 0.8āmgāmlā1 collagenase (Wako Pure Chemical Industries, Osaka, Japan) for 1āh. Whole-cell currents of the oocytes were recorded 3ā8 days after injection with a conventional two-electrode voltage clamp (Kobayashi et al, 1999; Ikeda et al, 2003). All recordings were carried out at room temperature (19Ā°C) to avoid damage to Xenopus oocytes and the effects of temperature (Fraser and Djamgoz, 1992; Weber, 1999). The membrane potential was held at ā70āmV unless otherwise specified. Microelectrodes were filled with 3āM KCl. The oocytes were placed in a 0.05āml narrow chamber and continuously superfused with a high-potassium (hK) solution (96āmM KCl, 2āmM NaCl, 1āmM MgCl2, 1.5āmM CaCl2, and 5āmM HEPES, pH 7.4 with KOH) or a K+-free high-sodium (ND98) solution (98āmM NaCl, 1āmM MgCl2, 1.5āmM CaCl2, and 5āmM HEPES, pH 7.4 with NaOH) at a flow rate of 2.5āml/min. In the hK solution, the K+ equilibrium potential was close to 0āmV, and the inward K+ current flow through the Kir channels was observed at negative holding potentials as shown earlier (Ho et al, 1993; Kubo et al, 1993a; Lesage et al, 1995; Kobayashi et al, 2006). Additionally, to examine the effects of the NRIs on outward K+ currents, a perfusion solution containing 4āmM K+ (K4 solution) was made by substituting NaCl with KCl in the ND98 solution. To examine the effects of the drugs on GIRK channels activated by G-protein activation, 13.8ānl of 100āmM Li4-guanosine-5ā²-O-(3-thiotriphosphate) (GTPĪ³S; Sigma-Aldrich), a nonhydrolyzable G-protein activator, dissolved in distilled water was injected into an oocyte using a nanoliter injector (World Precision Instruments, Sarasota, FL, USA) as described earlier (Kovoor et al, 1995). Furthermore, to examine the effects of intracellular atomoxetine, 23ānl of 10āmM atomoxetine dissolved in distilled water was injected into an oocyte using a nanoliter injector (Kobayashi et al, 2003), and the oocyte currents were then continuously recorded for ā¼30ā40āmin. As the volume of the Xenopus oocytes used was ā¼1āĪ¼l, the intracellular concentration of atomoxetine was presumed to be ā¼225āĪ¼M. For analysis of concentrationāresponse relationships, data were fitted to the following logistic equation: drug inhibition=max/1+(EC50/[drug])nH, with max being the maximal inhibition attainable, EC50 being the concentration of a drug that produces 50% of the maximal current response for that drug, [drug] being the concentration of an NRI and nH being the Hill coefficient, using KaleidaGraph (Synergy Software, Reading, PA, USA). The concentrations required to reduce control currents, by 25 and 50% (IC25 and IC50, respectively), were calculated from the concentrationāresponse relationships.
Statistical Analysis
Data are expressed as meanĀ±SEM, and n is the number of oocytes tested. Statistical analysis of the differences between groups was performed using Student's t-test, paired t-test, one-way analysis of variance (ANOVA), or two-way ANOVA followed by TukeyāKramer post hoc test. Values of P<0.05 were considered statistically significant.
Compounds
Tomoxetine hydrochloride (recently renamed atomoxetine hydrochloride) and reboxetine mesylate were purchased from Tocris Cookson (Bristol, UK) and dissolved in dimethyl sulfoxide (DMSO) or distilled water. The stock solution of each compound was stored at ā30Ā°C until use. Ethanol was purchased from Wako Pure Chemical Industries. Each compound was added to the perfusion solution in appropriate amounts immediately before the experiments.
RESULTS
Inhibition of GIRK Channels by Atomoxetine and Reboxetine
In Xenopus oocytes injected with GIRK1 and GIRK2 mRNAs, basal GIRK currents, which depend on free G-protein Ī²Ī³ subunits present in the oocytes because of the inherent activity of G-proteins (Dascal, 1997), were observed at a holding potential of ā70āmV in an hK solution containing 96āmM K+ (Figure 1a). Extracellular application of 30āĪ¼M atomoxetine or reboxetine reversibly reduced the inward currents through the expressed GIRK channels (Figure 1a). The current responses to an additional 100āĪ¼M atomoxetine during the application of 3āmM Ba2+, which blocks Kir channels, were not significant (reduction of inward currents by 5.5Ā±5.0ānA; <1% inhibition of the Ba2+-sensitive current components; n=4). The 3āmM Ba2+-sensitive current components (910.5Ā±65.7ānA, n=14) are considered to correspond to the magnitude of GIRK currents in oocytes expressing GIRK channels (Kobayashi et al, 1999). Atomoxetine and reboxetine produced no significant response in a K+-free ND98 perfusion solution containing 98āmM Na+ instead of the hK solution (n=4; data not shown), suggesting that the NRI-sensitive current components show K+ selectivity. Additionally, application of DMSO or distilled water, the solvent vehicle, at the highest concentration (0.3%) induced no significant current response in the hK or ND98 solutions (n=5; data not shown). However, in oocytes injected with mRNA for Kir1.1, an ATP-regulated Kir channel (Ho et al, 1993), or Kir2.1, a constitutively active Kir channel (Kubo et al, 1993a), extracellular application of 300āĪ¼M atomoxetine or reboxetine had no significant effects on the inward currents through the channels in the hK solution (<3% change of the Ba2+-sensitive current components; 583.3Ā±59.7ānA for Kir1.1, n=4; 1306.7Ā±179.8ānA for Kir2.1, n=4; Figure 1b). In uninjected oocytes, 300āĪ¼M atomoxetine and reboxetine as well as 3āmM Ba2+ caused no significant response (3.8Ā±2.9, 0Ā±0, and 6.8Ā±0.7ānA, respectively; n=4, 4, and 7, respectively; Figure 1c) compared with oocytes injected with GIRK mRNA, suggesting no significant effects of atomoxetine, reboxetine, or Ba2+ on intrinsic oocyte channels. Furthermore, in oocytes injected with GIRK1 and GIRK2 mRNAs, outward currents observed at a holding potential of ā30āmV in a K4 solution containing 4āmM K+ were reversibly reduced by 30āĪ¼M atomoxetine (n=4), 30āĪ¼M reboxetine (n=4), or 3āmM Ba2+ (the Ba2+-sensitive current components, 85.2Ā±32.8ānA, n=8; Supplementary Figure S2), whereas in uninjected oocytes, the NRIs at 100āĪ¼M and 3āmM Ba2+ caused no significant response (3.0Ā±0.9ānA for atomoxetine, 0Ā±0ānA for reboxetine, and 7.6Ā±1.3ānA for Ba2+; n=4, 4, and 8, respectively). The results suggest that the NRIs also inhibited outward GIRK currents. Similarly, in oocytes injected with either GIRK1 and GIRK4 mRNAs or GIRK2 mRNA, atomoxetine and reboxetine inhibited basal GIRK currents under the same conditions (3āmM Ba2+-sensitive current components for GIRK1/4, 1027.5Ā±112.6ānA, n=10; 3āmM Ba2+-sensitive current components for GIRK2, 757.0Ā±51.5ānA, n=12; Figure 2). The results suggest that atomoxetine and reboxetine inhibited GIRK channels, but not Kir1.1 and Kir2.1 channels.
Characteristics of Inhibition of GIRK Channels by Atomoxetine and Reboxetine
The concentrationāresponse relationships of the inhibitory effects of atomoxetine and reboxetine on GIRK1/2, GIRK2, and GIRK1/4 channels were investigated. Figure 2 shows that inhibition of these types of GIRK channels by atomoxetine and reboxetine was concentration-dependent. Table 1 shows the EC50 and nH values obtained from the concentrationāresponse relationships and the percentage inhibition of the GIRK currents by the NRIs at the highest concentrations tested. Additionally, because the drugs could not completely block these types of GIRK channels, even at the highest concentrations tested, the IC25 and IC50 values were also calculated to further compare the effects of the drugs (Table 1). The inhibition of GIRK1/4 channels by atomoxetine was more effective at 10 and 30āĪ¼M than inhibition of GIRK2 channels (P<0.05, TukeyāKramer post hoc test), although the effects of atomoxetine at the highest concentration on three types of channels were similar (P>0.05, TukeyāKramer post hoc test; Figure 2a; Table 1). In contrast, the inhibitory effects of reboxetine on these types of channels were statistically similar (P>0.05 at each concentration, TukeyāKramer post hoc test), although the inhibition of GIRK2 channels by 100 and 300āĪ¼M reboxetine was slightly less effective than inhibition of the other channel types (Figure 2b). Furthermore, inhibition of GIRK1/4 channels by 10āĪ¼M atomoxetine was more effective than 10āĪ¼M reboxetine (P<0.05, TukeyāKramer post hoc test), whereas the effects of atomoxetine on GIRK1/2 and GIRK2 channels were similar to reboxetine (P>0.05 at each concentration, TukeyāKramer post hoc test).
Instantaneous GIRK1/2 currents elicited by the voltage step to ā100āmV from a holding potential of 0āmV were diminished in the presence of 30āĪ¼M atomoxetine applied for 3āmin (Figure 3a). The percentage inhibition of the steady-state GIRK current at the end of the voltage step by atomoxetine was not significantly different from that of the instantaneous current (P>0.05, paired t-test; n=9 at ā40, ā60, ā80, ā100, and ā120āmV, respectively). For reboxetine, similar results were observed (n=7). These results suggest that the channels were inhibited by atomoxetine and reboxetine primarily at the holding potential of 0āmV and time-independently during each voltage pulse. Similar to the 3āmM Ba2+-sensitive current components corresponding to the magnitudes of basal GIRK currents, the magnitudes of currents reduced by 30āĪ¼M atomoxetine in oocytes expressing GIRK1/2 channels increased with negative membrane potentials, and the currentāvoltage relationships showed strong inward rectification (n=9; Figure 3b), indicating a characteristic of GIRK currents. The percentage inhibition of GIRK1/2 currents by 30āĪ¼M atomoxetine showed no significant difference across voltages between ā120 and ā40āmV (no significant atomoxetine effect Ć membrane potential effect interaction, P>0.1, one-way ANOVA; P>0.1 across voltages, TukeyāKramer post hoc test; Figure 3c). For reboxetine, similar results were observed (n=7; Figure 3b and c). The results suggest that the inhibition of GIRK channels by atomoxetine and reboxetine was voltage-independent. Furthermore, similar results were obtained in oocytes expressing GIRK1/4 channels (n=5 for atomoxetine and n=4 for reboxetine; data not shown). Therefore, atomoxetine and reboxetine may have similar actions as GIRK channel inhibitors.
Atomoxetine possesses a secondary amine group with a pKa value of 9.23 (Eli Lilly and Company Data Sheet; Supplementary Figure S1). At physiological pH or below, atomoxetine exists mainly in a protonated form, ā¼98.5% at pH 7.4, and the proportion of the uncharged form increases with an increase in pH. We examined whether changes in extracellular pH would affect GIRK channel inhibition by atomoxetine. However, in oocytes expressing GIRK1/2 channels, the percentage inhibition of GIRK channels by atomoxetine at the same concentrations was not significantly affected by extracellular pH 7.4 and 9.2 (no significant pH Ć atomoxetine interaction, P>0.5, two-way ANOVA; P>0.1 at each concentration, TukeyāKramer post hoc test; Figure 4). The results indicate that a marked increase in the proportion of the uncharged form may not significantly affect all of the effects on GIRK channels, suggesting that GIRK channel inhibition may be mediated by both forms of atomoxetine with similar effectiveness.
Effects of Atomoxetine and Reboxetine on GIRK Channels Activated by a G-Protein-Coupled Receptor or GTPĪ³S
We examined the effects of atomoxetine and reboxetine on GIRK channels activated by a G-protein-coupled receptor. In oocytes co-expressing GIRK1/2 channels and A1Rs (Kobayashi et al, 2002), 100ānM adenosine significantly induced inward GIRK currents (1000.7Ā±76.9ānA, n=10; Figure 5a), and 300āĪ¼M atomoxetine or reboxetine alone consistently inhibited basal GIRK currents (3āmM Ba2+-sensitive current components, 157.2Ā±31.3ānA, n=10). The current responses to 100ānM adenosine were reduced by the addition of atomoxetine or reboxetine (n=5 for each NRI; Figure 5a). These results suggest that atomoxetine and reboxetine inhibited total GIRK currents through the GIRK channels activated by the A1R and the basally active GIRK channels. The percentage inhibition of total GIRK currents by atomoxetine or reboxetine (IC25=4.5Ā±1.6 and 8.6Ā±1.7āĪ¼M; IC50=42.7Ā±12.3 and 55.1Ā±16.4āĪ¼M; nH=0.93Ā±0.04 and 0.79Ā±0.13; n=5, respectively; Figure 5b) was not significantly different from that of basal GIRK currents in oocytes injected with GIRK1 and GIRK2 mRNAs (P>0.05, IC25 and IC50 values for each NRI, Student's t-test; P>0.05 at each concentration, TukeyāKramer post hoc test), suggesting that the effects of the NRIs on A1R-activated GIRK channels were similar to those on GIRK channels activated by basally free G-protein Ī²Ī³ subunits present in oocytes.
GIRK channels are activated by various G-protein-coupled receptors through the direct action of G-protein Ī²Ī³ subunits released from the heterotrimeric G-protein complex (Dascal, 1997; Kobayashi and Ikeda, 2006). The effects of the NRIs on GIRK channels activated by G-protein-coupled signaling mechanisms were further examined using GTPĪ³S, a nonhydrolyzable GTP analogue that maintains G-proteins in an activated state. Injection of GTPĪ³S into Xenopus oocytes injected with GIRK1 and GIRK2 mRNAs increased inward currents with time and reached a steady-state level (938.9Ā±119.2ānA, n=18) as reported earlier (Kovoor et al, 1995). The increased inward currents were completely blocked by 3āmM Ba2+, whereas GTPĪ³S injection into uninjected oocytes had no significant effect on current responses to 3āmM Ba2+ (3.9Ā±2.1ānA, n=9). Increased GIRK currents composed of basal GIRK currents and GTPĪ³S-induced GIRK currents were inhibited by atomoxetine or reboxetine (IC50=29.0Ā±6.2 and 52.3Ā±10.1āĪ¼M; nH=1.28Ā±0.04 and 1.14Ā±0.06; n=6 and 12, respectively). The percentage inhibition of total GIRK currents by atomoxetine or reboxetine was not significantly different from that of basal GIRK currents in GTPĪ³S-untreated oocytes injected with GIRK1 and GIRK2 mRNAs (P>0.05, IC50 value for each NRI, Student's t-test; P>0.05 at each concentration, TukeyāKramer post hoc test), suggesting that the effects of the NRIs on basally active GIRK channels and GIRK channels activated by G-protein activation induced by GTPĪ³S were similar.
Atomoxetine Inhibits Ethanol-Induced GIRK Currents
GIRK channels are also activated by ethanol independent of G-protein signaling pathways (Kobayashi et al, 1999). Atomoxetine was shown to reduce cumulative heavy drinking days in the treatment of psychiatric patients with comorbid alcohol use disorders (Wilens et al, 2008). Therefore, we also examined the effects of atomoxetine on GIRK channel activation induced by ethanol. The effects of atomoxetine were evaluated by measuring the amplitude of the ethanol-induced current response during extracellular application of atomoxetine at different concentrations. In oocytes injected with GIRK1 and GIRK2 mRNAs, the GIRK currents induced by 100āmM ethanol (420.0Ā±32.5ānA, n=5) were reversibly attenuated in the presence of atomoxetine (IC25=5.8Ā±1.1āĪ¼M; IC50=15.4Ā±3.1āĪ¼M; nH=1.22Ā±0.22; n=5; Figure 6a and b). However, 100āmM ethanol-induced GIRK currents were not significantly affected by intracellularly applied atomoxetine (104.3Ā±2.8% of untreated control current, paired t-test, P>0.1, n=5; Figure 6c). Moreover, in oocytes expressing GIRK channels, the basal currents were not significantly affected by intracellularly applied atomoxetine (103.0Ā±2.2% of untreated control current, paired t-test, P>0.1, n=5). The results indicate that intracellular atomoxetine could not inhibit GIRK channels. In contrast, GIRK channel inhibition induced by extracellularly applied atomoxetine, which is mainly protonated at pH 7.4, was reversible with washout (Figures 1a and 6a). As the protonated form may not readily permeate the cell membrane, extracellularly applied atomoxetine may exist mainly on the extracellular side. Altogether, extracellular atomoxetine may inhibit GIRK channels activated by ethanol.
DISCUSSION
In this study, we showed that atomoxetine and structurally related reboxetine, clinically used selective NRIs, inhibited brain-type GIRK1/2 and GIRK2 channels and cardiac-type GIRK1/4 channels expressed in Xenopus oocytes. However, Kir1.1 and Kir2.1 channels in other Kir channel subfamilies were insensitive to both NRIs. The inhibitory effects on GIRK channels were concentration-dependent, but voltage-independent, and time-independent during each voltage pulse. The present results suggest that the site of action on the channels may be extracellular. In contrast, blockade of GIRK channels by extracellular Ba2+ and Cs+, which occlude the pore of the open channel, shows a concentration-dependence, a voltage-dependence, and a time-dependence with a comparatively small effect on the instantaneous current but a marked inhibition of the steady-state current at the end of the voltage pulses (Lesage et al, 1995). These observations suggest that atomoxetine and reboxetine may cause an allosteric conformational change in GIRK channels even before the voltage pulses, rather than simple occlusion of the open channel. Interestingly, GIRK channels are also inhibited by the selective serotonin reuptake inhibitor (SSRI) fluoxetine (Kobayashi et al, 2003; Takahashi et al, 2006), despite a great difference in the pharmacological profiles for monoamine transporters between the two NRIs and fluoxetine. The chemical structures of atomoxetine and reboxetine are related to fluoxetine (Boot et al, 2005; Supplementary Figure S1), suggesting that the common moiety of the structures may play a key role in interacting with GIRK channels. Additionally, the Xenopus oocyte expression assay with a conventional two-electrode voltage clamp is generally conducted using defolliculated oocytes, which are still covered over the plasma membrane with the vitelline membrane, at room temperature (Fraser and Djamgoz, 1992; Weber, 1999; Ikeda et al, 2003). Further studies using mammalian cells, including neurons and cardiac myocytes, at physiological temperature may be useful for advancing our understanding of the effects of NRIs on GIRK channels.
Atomoxetine is predominantly metabolized by the genetically polymorphic cytochrome P450 2D6 (CYP2D6) pathway, and its pharmacokinetics and metabolism are characterized by two distinct activities of CYP2D6: active or poor metabolic capability (Witcher et al, 2003; Simpson and Plosker, 2004). The maximum plasma concentrations during treatment with atomoxetine at therapeutic doses ranged from ā¼0.7ā4.8āĪ¼M in CYP2D6 active metabolizers (Witcher et al, 2003), whereas those in CYP2D6 poor metabolizers (ā¼7% of the Caucasian population) were six-fold higher than those in CYP2D6 active metabolizers (Simpson and Plosker, 2004). Additionally, co-administration of the SSRI paroxetine, a potent inhibitor of CYP2D6, increased the plasma concentrations of atomoxetine by 3.5-fold, with a pharmacokinetic profile similar to CYP2D6 poor metabolizers (Belle et al, 2002), suggesting a significant increase in atomoxetine concentrations with concomitant treatment with CYP2D6 inhibitors. The maximum plasma concentrations of reboxetine at therapeutic doses in depressed patients ranged from 0.5 to 2.1āĪ¼M (Poggesi et al, 2000). Additionally, increases in doses of the NRIs are associated with increases in plasma concentrations (Ćhman et al, 2001; Witcher et al, 2003), and the concentration in a fatal case of atomoxetine overdose was reported to be up to 32.5āĪ¼M (Garside et al, 2006). Recent studies using radiolabeled NRI ligands have indicated that NRIs are extensively distributed in most tissues (Kiyono et al, 2004, 2008; Kanegawa et al, 2006). Indeed, brain and heart levels of NRIs were ā¼4.7- to 6.5-fold and 9- to 12-fold higher for atomoxetine (Kiyono et al, 2004) and ā¼15- to 16-fold and 21- to 32-fold higher for reboxetine than corresponding blood levels, respectively (Kanegawa et al, 2006; Kiyono et al, 2008). Therefore, brain and heart concentrations during treatment with therapeutic doses of atomoxetine and reboxetine, as well as after overdose, overlap with their effective concentrations in inhibiting brain- and cardiac-type GIRK channels (Figure 2). GIRK channels in the brain and heart may be inhibited by atomoxetine and reboxetine, particularly with the use of atomoxetine with CYP2D6 poor metabolizers or co-administration of CYP2D6 inhibitors. Inhibition of GIRK channels causes a depolarization of membrane potential, resulting in an increase in cell excitability (Kuzhikandathil and Oxford, 2002). GIRK channels have an important function in regulating neuronal excitability, synaptic transmission, and heart rate (LĆ¼scher et al, 1997; Kovoor et al, 2001). Therefore, even partial inhibition of GIRK channels by atomoxetine and reboxetine may affect certain brain and heart functions.
Interestingly, GIRK2 knockout mice exhibit reduced anxiety-related behavior (Blednov et al, 2001). In clinical studies, reboxetine and atomoxetine were effective in the treatment of panic disorder and comorbid anxiety disorder, respectively (Versiani et al, 2002; Geller et al, 2007), suggesting their anxiolytic properties. Although their therapeutic effects are generally thought to be primarily attributable to inhibition of norepinephrine reuptake in the brain (HajĆ³s et al, 2004; Simpson and Plosker, 2004), inhibition of GIRK channels may also contribute to improvement of anxiety symptoms.
GIRK2 knockout mice exhibit spontaneous seizures and are more susceptible to seizures induced by pentylenetetrazol than wild-type mice (Signorini et al, 1997). In animal studies using atomoxetine or reboxetine, convulsions were observed only at extremely high doses (Wong et al, 2000; Wernicke et al, 2007). The incidence of seizures during treatment with NRIs has been reportedly rare (Montgomery, 2005; Wernicke et al, 2007). Brain levels of the drugs in overdose cases may be considerably higher than levels during treatment at therapeutic doses (Poggesi et al, 2000; Kiyono et al, 2004, 2008; Garside et al, 2006; Kanegawa et al, 2006), suggesting that potent inhibition of neuronal GIRK channels by atomoxetine and reboxetine after overdose may contribute to increased seizure activity. However, the NRIs simultaneously increase extracellular levels of norepinephrine in the brain (HajĆ³s et al, 2004; Simpson and Plosker, 2004), and norepinephrine has anticonvulsant effects (Ahern et al, 2006). The enhancement of norepinephrine by NRIs may be involved in the rare incidence of seizures. Although atomoxetine and reboxetine are generally well tolerated and have a benign side effect profile (HajĆ³s et al, 2004; Simpson and Plosker, 2004), the inhibitory effects on GIRK channels may be partly related to the occurrence of other neurological side effects, such as insomnia and dizziness.
In the heart, GIRK channels cause slowing of heart rate in response to activation of M2 muscarinic receptors through acetylcholine release from the stimulated vagus nerve (Kubo et al, 1993b; Krapivinsky et al, 1995). GIRK1 or GIRK4 knockout mice exhibit slightly elevated resting heart rates (Bettahi et al, 2002). Atomoxetine and reboxetine are associated with modest increases in heart rate (HajĆ³s et al, 2004; Simpson and Plosker, 2004) and tachycardia in cases of toxicity (LoVecchio and Kashani, 2006). The binding affinities of atomoxetine and reboxetine for the muscarinic receptor are in the low micromolar range (Cusack et al, 1994; Wong et al, 2000; HajĆ³s et al, 2004). Inhibition of norepinephrine reuptake enhances sympathetic nerve activity (Keller et al, 2004). The present results indicate that atomoxetine and reboxetine inhibit cardiac-type GIRK1/4 channels at clinically relevant heart concentrations. Altogether, an increase in heart rate during treatment with the drugs may be related to not only enhancement of sympathetic nerve activity and antagonism of the muscarinic receptor but also inhibition of atrial GIRK channels. Additionally, QT interval prolongation in two cases with atomoxetine overdose was reported (Barker et al, 2004; Sawant and Daviss, 2004). Recently, atomoxetine at micromolar concentrations was shown to inhibit cloned human ether-a-go-go-related gene (hERG) channels underlying rapidly activating delayed rectifier K+ currents using the Xenopus oocyte expression assay (Scherer et al, 2009). Inhibition of delayed rectifier K+ currents induces QT prolongation (Scherer et al, 2009), and QT prolongation after atomoxetine overdose may be related to inhibition of hERG channels but not GIRK channels among cardiac K+ channels. Furthermore, GIRK4 knockout mice are resistant to atrial fibrillation caused by vagal stimulation without showing any changes in atrioventricular nodal function and ventricular arrhythmias (Kovoor et al, 2001). Tertiapine, a selective GIRK blocker in the heart, terminates atrial fibrillation, the most common arrhythmia (Hashimoto et al, 2006). Atomoxetine and reboxetine may therefore have an advantage in treating psychiatric patients with comorbid atrial fibrillation.
Atomoxetine was shown to reduce cumulative heavy drinking days in the treatment of psychiatric patients with comorbid alcohol use disorders (Wilens et al, 2008). Interestingly, GIRK2 knockout mice show reduced ethanol-induced conditioned taste aversion and conditioned place preference and are less sensitive than wild-type mice to some of the acute effects of ethanol, including anxiolysis, habituated locomotor stimulation, and acute handling-induced convulsions (Hill et al, 2003). In the present study, atomoxetine inhibited ethanol-induced GIRK1/2 currents, suggesting that it may suppress some GIRK-related effects of ethanol. Furthermore, GIRK knockout mice also show reduced cocaine self-administration (Morgan et al, 2003) and attenuation of the morphine withdrawal syndrome (Cruz et al, 2008). In the nervous system, GIRK channels are activated by Ī¼-opioid and CB1 cannabinoid receptors (North, 1989; Dascal, 1997; Kobayashi and Ikeda, 2006). Reboxetine and atomoxetine have also been shown to be useful in the treatment of cocaine dependence and marijuana users, respectively (Tirado et al, 2008; Szerman et al, 2005). Inhibition of GIRK channels by atomoxetine and reboxetine may have a role in the treatment of drug addiction.
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Acknowledgements
We are grateful to Dr Kansaku Baba for his cooperation and Mr Kazuo Kobayashi (Niigata University) for his assistance. We also thank Dr Steven C Hebert and Dr Lily Y Jan for generously providing the Kir1.1 cDNA and Kir2.1 cDNA, respectively. This work was supported by research grants from the Ministry of Education, Science, Sports, and Culture of Japan and from the Ministry of Health, Labour, and Welfare of Japan.
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The authors declare that over the past 3 years Kazutaka Ikeda has received research grants or expenses that are not related to this study from Fujifilm Corporation, the Mitsubishi Foundation, the Naito Foundation, and the Smoking Science Foundation, and a lecture fee from Dainippon Sumitomo Pharma and Kyowa Hakko Kirin. The authors declare that, except for income received from their primary employer and the aforementioned disclosures, no financial support or compensation has been received from any individual or corporate entity over the past 3 years for research or professional service, and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.
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Kobayashi, T., Washiyama, K. & Ikeda, K. Inhibition of G-Protein-Activated Inwardly Rectifying K+ Channels by the Selective Norepinephrine Reuptake Inhibitors Atomoxetine and Reboxetine. Neuropsychopharmacol 35, 1560ā1569 (2010). https://doi.org/10.1038/npp.2010.27
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DOI: https://doi.org/10.1038/npp.2010.27
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