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

Figures

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.