Korean J Physiol Pharmacol.  2018 Sep;22(5):585-595. 10.4196/kjpp.2018.22.5.585.

Tricyclic antidepressant amitriptyline inhibits 5-hydroxytryptamine 3 receptor currents in NCB-20 cells

• Park YS
• Myeong SH
• Kim IB
• Sung KW

• KMID: 2419008
• DOI: http://doi.org/10.4196/kjpp.2018.22.5.585

Abstract

Amitriptyline, a tricyclic antidepressant, is commonly used to treat depression and neuropathic pain, but its mechanism is still unclear. We tested the effect of amitriptyline on 5-hydroxytryptamine 3 (5-HT₃) receptor currents and studied its blocking mechanism because the clinical applications of amitriptyline overlapped with 5-HT₃ receptor therapeutic potentials. Using a whole-cell voltage clamp method, we recorded the currents of the 5-HT₃ receptor when 5-HT was applied alone or co-applied with amitriptyline in cultured NCB-20 neuroblastoma cells known to express 5-HT₃ receptors. To elucidate the mechanism of amitriptyline, we simulated the 5-HT₃ receptor currents using Berkeley Madonna® software and calculated the rate constants of the agonist binding and receptor transition steps. The 5-HT₃ receptor currents were inhibited by amitriptyline in a concentration-dependent, voltage-independent manner, and a competitive mode. Amitriptyline accelerated the desensitization of the 5-HT₃ receptor. When amitriptyline was applied before 5-HT treatment, the currents rose slowly until the end of 5-HT treatment. When amitriptyline was co-applied with 5-HT, currents rose and decayed rapidly. Peak current amplitudes were decreased in both applications. All macroscopic currents recorded in whole cell voltage clamping experiments were reproduced by simulation and the changes of rate constants by amitriptyline were correlated with macroscopic current recording data. These results suggest that amitriptyline blocks the 5-HT₃ receptor by close and open state blocking mechanisms, in a competitive manner. We could expand an understanding of pharmacological mechanisms of amitriptyline related to the modulation of a 5-HT₃ receptor, a potential target of neurologic and psychiatric diseases through this study.

Keyword

5-Hydroxytriptamine 3 receptor; Amitriptyline; Depression; Patch clamp; Simulation

MeSH Terms

Amitriptyline*
Constriction
Depression
Methods
Neuralgia
Neuroblastoma
Serotonin*
Amitriptyline
Serotonin

Figure

• 

  Fig. 1 Characteristics of 5-HT3 receptor currents in NCB-20 neuroblastoma cells. (A) Representative 5-HT3 receptor currents induced by 0.3, 1, 3, 10, 30 µM of 5-HT. 5-HT was applied for 5 s indicated by the open horizontal bar. 5-HT3 receptor current amplitudes were increased depending on 5-HT concentrations. (B) Averaged concentration-response curve of 5-HT3 receptor currents. Data were normalized to the peak amplitude induced by 10 µM of 5-HT, which was taken as 1. EC50 value was 1.73±0.08 µM with Hill coefficient of 2.44±0.08 (n=10). Data are expressed as mean±S.E.M.

• 

  Fig. 2 Concentration-dependent inhibition of 5-HT3 receptor currents by amitriptyline. (A) Representative current traces induced by 5-HT 10 µM (open horizontal bar) co-applied with 0.3, 1, 3, 10, 30 µM of amitriptyline (closed horizontal bar). (B) Representative current traces induced by 5-HT 3 µM (open horizontal bar) co-applied with 0.3, 1, 3, 10, 30 µM of amitriptyline (closed horizontal bar). Inhibitory effect of the amitriptyline was profound at the 3 µM of 5-HT induced currents. (C) Averaged concentration-inhibition curve of 5-HT3 receptor currents by amitriptyline. Data were normalized to the peak amplitude induced by 10 µM of 5-HT (○) or 3 µM of 5-HT (●). IC50 of amitriptyline on 3 µM 5-HT-induced currents was 1.78±0.37µM with Hill coefficient of 1.00±0.08 (n=10 cells), and IC50 of amitriptyline on 10 µM 5-HT was 6.36±0.4µM with Hill coefficient of 0.91±0.04 (n=10 cells). IC50 of amitriptyline on 3 µM 5-HT was significantly lower than that of 10 µM 5-HT (unpaired t-test, p<0.001). Data are expressed as mean±S.E.M.

• 

  Fig. 3 Change of 5-HT concentration-response relationship by amitriptyline. (A–E) Representative traces of 5-HT3 receptor currents induced by 0.3, 1, 3, 10, 30 µM of 5-HT (open horizontal bar) in the presence (indicated by arrow) or absence of 3 µM amitriptyline (closed horizontal bar). Amitriptyline inhibited 5-HT induced currents more effectively at the lower concentration of 5-HT. (F) Averaged concentration-response curve of 5-HT3 receptor currents in the presence (●) or absence (○) of amitriptyline. Data were normalized to the peak amplitude induced by 10 µM of 5-HT. EC50 of 5-HT was increased from 1.73±0.08 µM to 9.21±0.06 µM by amitriptyline (p<0.001, n=10). Amitriptyline did not decrease the peak amplitudes induced by 30 µM of 5-HT (paired t-test, p=0.13, n=7). Data are expressed as mean±S.E.M.

• 

  Fig. 4 Effect of amitriptyline on current-voltage (I-V) relationship of 5-HT3 receptor currents. (A) Representative traces of 5-HT3 receptor currents induced by 3 µM of 5-HT alone at the holding potentials of −50−+30 mV. (B) 5-HT3 receptor currents induced by 3 µM of 5-HT at the holding potentials of −50−+30 mV with amitriptyline 3 µM. (C) I-V plots of averaged peak amplitude induced by 5-HT with (●) or without (○) amitriptyline (AMT). The reversal potentials were 8.54±0.32 mV at 5-HT alone, and 8.88±0.44 mV at 5-HT with amitriptyline. Reversal potential was not significantly changed by co-application of amitriptyline (p=0.4899, n=10). (D) Fractional block of amitriptyline (peak amplitude of 5-HT with amitriptyline/peak amplitude of 5-HT alone) as a function of holding potentials. Inhibitory effect of amitriptyline showed a voltage independency. Data are expressed as mean±S.E.M.

• 

  Fig. 5 5-HT3 receptor currents induced by different modes of amitriptyline application. (A) Representative trace of 5-HT3 receptor currents induced by 5-HT (3 µM) alone (open horizontal bar) and co-application of 5-HT and amitriptyline (3 µM, closed horizontal bar, indicated by arrow). (B) Representative traces of 5-HT3 receptor currents induced by 5-HT alone and pre-application of amitriptyline for 10 s (indicated by arrow). (C) Representative traces of 5-HT3 receptor currents induced by 5-HT alone and co-application of 5-HT and amitriptyline after 10 s preapplication of amitriptyline (indicated by arrow). (D) Averaged inhibitory ratio (peak amplitude of 5-HT with amitriptyline/peak amplitude of 5-HT alone) at each application mode of amitriptyline. 5-HT induced current was blocked by amitriptyline in both of pre-application (Pre) and co-application (Co) modes, and completely blocked by combination of pre- and co-application (Pre & Co). Data are expressed as mean±S.E.M.

• 

  Fig. 6 Effect of amitriptyline on desensitization of 5-HT3 receptor. (A) Representative 5-HT3 receptor currents induced by 10 µM of 5-HT (open horizontal bar) for 15 s with or without 3 µM of amitriptyline (closed horizontal bar, indicated by arrow). (B) Averaged desensitization time constant of the 5-HT alone (5-HT) and with amitriptyline (5-HT+AMT). Amitriptyline decreased the time constant of 5-HT3 receptor current decay (paired t-test, *p<0.01, n=9) suggesting the desensitization was accelerated by amitriptyline. Data are expressed as mean±S.E.M.

• 

  Fig. 7 Kinetic schemes and simulated 5-HT3 receptor currents. (A) Ion channel kinetic scheme of 5-HT3 receptor (R), 5-HT (A), and amitriptyline (B) in each application mode, 5-HT alone, co-application of amitriptyline with 5-HT (Co-app), and pre-application of amitriptyline with 5-HT (Pre-app). (B) Recorded current trace (gray) and simulated 5-HT current (black) in each application mode. The simulated 5-HT current was well matched with the recorded current trace.

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