Korean J Physiol Pharmacol 2017;21(2):225-232 https://doi.org/10.4196/kjpp.2017.21.2.225 Original Article Nortriptyline, a tricyclic antidepressant, inhibits voltage- dependent K+ channels in coronary arterial smooth muscle cells Sung Eun Shin1,#, Hongliang Li1,#, Han Sol Kim1, Hye Won Kim1, Mi Seon Seo1, Kwon-Soo Ha2, Eun-Taek Han3, Seok-Ho Hong4, Amy L. Firth5, Il-Whan Choi6, Young Min Bae7, and Won Sun Park1,* Departments of 1Physiology, 2Molecular and Cellular Biochemistry, 3Medical Environmental Biology and Tropical Medicine, 4Internal Medicine, Kangwon National University School of Medicine, Chuncheon 24341, Korea, 5Department of Pulmonary, Critical Care and Sleep Medicine, University of Southern California, Keck School of Medicine, Los Angeles, CA90033, USA, 6Department of Microbiology, Inje University College of Medicine, Busan 48516, Korea, 7Department of Physiology, Konkuk University School of Medicine, Chungju 27478, Korea ARTICLE INFO ABSTRACT We demonstrated the effect of nortriptyline, a tricyclic antidepressant Received November 14, 2016 + Revised December 7, 2016 drug and serotonin reuptake inhibitor, on voltage-dependent K (Kv) channels in Accepted December 7, 2016 freshly isolated rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Nortriptyline inhibited Kv currents in a concentration-dependent *Correspondence manner, with an apparent IC50 value of 2.86±0.52 mM and a Hill coefficient of Won Sun Park 0.77±0.1. Although application of nortriptyline did not change the activation curve, E-mail: [email protected] nortriptyline shifted the inactivation current toward a more negative potential. Application of train pulses (1 or 2 Hz) did not change the nortriptyline-induced Key Words Kv channel inhibition, suggesting that the effects of nortiprtyline were not use- Coronary artery dependent. Preincubation with the Kv1.5 and Kv2.1/2.2 inhibitors, DPO-1 and Nortriptyline + guangxitoxin did not affect nortriptyline inhibition of Kv channels. From these Voltage-dependent K channel results, we concluded that nortriptyline inhibited Kv channels in a concentration- #These authors contributed equally to dependent and state-independent manner independently of serotonin reuptake. this work. Introduction The alteration of membrane potential is regarded as a major determinant of changes in vascular diameter, and can thereby Tricyclic antidepressants (TCAs), including amitriptyline, alter blood pressure and organ blood flow [5,6]. Several ion butriptyline, nortriptyline, clomipramine, desipramine, and channels expressed in arterial smooth muscle, including Ca2+, doxepin, have been the first selection for pharmacological K+, and Cl– channels, are involved in maintaining and changing treatment of clinical depression for many years [1,2]. Nor- the membrane potential. Of these, K+ channels play the most triptyline is second-generation TCA and is mainly used in the crucial role in determining the resting membrane potential of treatment of major depression and childhood nocturnal enuresis arterial muscle cells. In arterial muscle cells, four types of major + + [3]. Additionally, although nortriptyline has been shown to have K channels have been detected: ATP-sensitive K (KATP), large- 2+ + + fewer side effects than tertiary amine TCAs, such as amitriptyline, conductance Ca -activated K (BKCa), inwardly rectifying K imipramine, and clomipramine, some side effects, including (Kir), and voltage-dependent K+ (Kv) channels [5,7]. Although dry mouth, sedation, constipation, mild blurred vision, tinnitus, most K+ channels play essential roles in maintaining resting euphoria, and mania, have still been observed [4]. However, to membrane potential, Kv channels in particular are regarded date, the side effects of nortriptyline on vascular smooth muscle, as the most crucial channels for regulating resting membrane specifically ion channels, are unknown. potential [8]. In fact, inhibition of Kv channels in some arteries This is an Open Access article distributed under the terms Author contributions: S.E.S., H.L. and W.S.P. Conceived and designed of the Creative Commons Attribution Non-Commercial the experiments. S.E.S., H.L., H.S.K., H.W.K. and M.S.S. Performed the License, which permits unrestricted non-commercial use, distribution, and experiments. K.S.H., E.T.H. and S.H.H. Analyzed the data. I.W.C. and Y.M.B. reproduction in any medium, provided the original work is properly cited. Contributed reagents/materials/analysis tools. S.E.S., H.L., A.L.F. and W.S.P. Copyright © Korean J Physiol Pharmacol, pISSN 1226-4512, eISSN 2093-3827 Wrote the manuscript. www.kjpp.net 225 Korean J Physiol Pharmacol 2017;21(2):225-232 226 Shin SE et al induces strong membrane depolarization [9,10]. Additionally, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). reduction of Kv channel activity and/or expression has been identified in various metabolic and circulatory diseases [8,11-13]. Electrophysiological recordings and data analysis Furthermore, the modulation of vascular Kv channel is closely related to various intracellular protein kinases, such as protein Kv current were recorded in individual vascular smooth kinase C (PKC), protein kinase A (PKA), and protein kinase muscle cells using PatchPro software, NI-DAQ-7 digital interface G (PKG) [7]. Therefore, the unexpected effects of some agents (National Instruments, Union, CA, USA), and EPC-8 amplifier on vascular Kv channels must be clearly identified to reduce (Medical system Corp., Darmstadt, Germany). Borosilicate potentially toxic vascular effects. capillary glass pipettes (Clark Electromedical Instruments, In this study, we demonstrated the inhibitory effect of Pangbourne, UK) were pulled on a PP-830 puller (Narishige nortriptyline on Kv channels using native coronary arterial Scientific Instrument Laboratory, Tokyo, Japan) with a resulting smooth muscle from rabbits. We found that nortriptyline inhibits resistance of 3~4 MΩ. Kv channels in a concentration-dependent and state-independent Data analysis was completed using Origin 7.0 software fashion independently of serotonin reuptake inhibition. (Microcal Software, Inc., Northampton, MA, USA). The drug- channel interaction kinetics is described as a first-order blocking model [14]. The values for the half-maximal inhibitory (IC50) and Methods the slope value (n) were calculated from concentration-dependent data and fitted to the Hill equation: Single cell isolation n ƒ=1/{1+(IC50/[D]) } In accordance with the guidelines of the Committee for Ani- mal Experiments of Kangwon National University, male New where ƒ represents the fractional current inhibition (ƒ= 1-Idrug/ Zealand White rabbits (2.0~2.5 kg) were anesthetized using Icontrol) at each potential, and [D] represents drug concentration. pentobarbitone sodium (40 mg/kg) and heparin (120 U/kg) Channel activation was calculated from depolarizing from injected simultaneously into the ear vein. The chest was opened, –80 to +60 mV in 10-mV increment returning to –40 mV. The the heart separated and left descending coronary arteries were recorded tail currents were normalized to the maximal tail dissected and collected in normal Tyrode’s solution. To obtain current at each depolarizing step and fitted with the Boltzmann a suspension of single smooth muscle cells, the arteries were equation: digested in 1 ml Ca2+-free normal Tyrode’s solution containing papain (1.0 mg/mL), bovine serum albumin (BSA, 1.0 mg/mL), y=1/{1+exp(–(V–V1/2)/k)} and dithiothreitol (DTT, 1.0 mg/mL) for 24 min at 37oC. After 2+ this incubation step, the solution was replaced with 1 ml Ca - where V is the test potential, V1/2 represents the half-point of free normal Tyrode solution containing collagenase (2.8 mg/ activation, and k is the slope factor. mL), BSA, and DTT, for 22 minutes at 37oC. A suspension of Steady-state inactivation was calculated using a two-step single smooth muscle cells was obtained by gentle agitation of the voltage protocol from a test potential of +40 mV for 600 ms after digested arteries using a pasture pipette and kept at 4oC in Kraft- 7-s of preconditioning pulses applied at potentials ranging from Brühe (KB) solution. Isolated cells were preserved at 4oC for use –80 to +30 mV in the absence and presence of nortriptyline. in experiments within 6 h. The steady-state inactivation curve was calculated from another Boltzmann equation: Solutions and chemicals y=1/[1+exp {(V–V1/2)/k}] The composition (in mM) of normal Tyrode’s solution was: CaCl2, 1.8; NaCl, 135; NaH2PO4, 0.33; KCl, 5.4; HEPES, 5; MgCl2, where V is the potential for preconditioning pulses, V1/2 is the 0.5; glucose, 16.6; adjusted to pH 7.4 with NaOH. The composition potential of the mid-maximal of inactivation, and k represents (in mM) of KB solution was: KCl, 55; KOH, 50; KH2PO4,70; the slope value. taurine, 20; L-glutamate, 20; MgCl2, 3; HEPES, 10; glucose, 18; All data is expressed as means±standard error of the mean EGTA, 0.6; adjusted to pH 7.3 with KOH. The composition (in (S.E.M) and the Student’s t-test was applied to determine sig- mM) of the pipette solution was: KCl, 25; K-aspartate, 110; NaCl, nificance with p<0.05 regarded as statistically significant. 5; Mg-ATP, 4; MgCl2, 2; HEPES, 10; EGTA, 10; adjusted to pH 7.2 with KOH. Nortriptyline was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and dissolved in distilled water. DPO-1 and guangxitoxin were purchased from Tocris Cookson (Ellisville, Korean J Physiol Pharmacol 2017;21(2):225-232 https://doi.org/10.4196/kjpp.2017.21.2.225 Nortriptyline inhibits Kv channels 227 Results Nortriptyline inhibits the Kv current in a dose- dependent manner Inhibition of Kv current by nortriptyline Several concentrations (0, 0.1, 0.3, 1, 3, 10, 30, and 100 mM) The inhibitory effects of nortriptyline were recorded using of nortriptyline were applied to determine whether the nor- native coronary smooth muscle cells from rabbit heart. The triptyline-induced inhibition of Kv channels was dose-dependent.