Korean J Physiol Pharmacol.  2019 Nov;23(6):509-517. 10.4196/kjpp.2019.23.6.509.

Selective serotonin reuptake inhibitor escitalopram inhibits 5-HT₃ receptor currents in NCB-20 cells

• Park YS
• Sung KW

• KMID: 2461044
• DOI: http://doi.org/10.4196/kjpp.2019.23.6.509

Abstract

Escitalopram is one of selective serotonin reuptake inhibitor antidepressants. As an S-enantiomer of citalopram, it shows better therapeutic outcome in depression and anxiety disorder treatment because it has higher selectivity for serotonin reuptake transporter than citalopram. The objective of this study was to determine the direct inhibitory effect of escitalopram on 5-hydroxytryptamine type 3 (5-HT₃) receptor currents and study its blocking mechanism to explore additional pharmacological effects of escitalopram through 5-HT₃ receptors. Using a whole-cell voltage clamp method, we recorded currents of 5-HT₃ receptors when 5-HT was applied alone or co-applied with escitalopram in cultured NCB-20 neuroblastoma cells known to express 5-HT₃ receptors. 5-HT induced currents were inhibited by escitalopram in a concentration-dependent manner. EC50 of 5-HT on 5-HT₃ receptor currents was increased by escitalopram while the maximal peak amplitude was reduced by escitalopram. The inhibitory effect of escitalopram was voltage independent. Escitalopram worked more effectively when it was co-applied with 5-HT than pre-application of escitalopram. Moreover, escitalopram showed fast association and dissociation to the open state of 5-HT₃ receptor channel with accelerating receptor desensitization. Although escitalopram accelerated 5-HT₃ receptor desensitization, it did not change the time course of desensitization recovery. These results suggest that escitalopram can inhibit 5-HT₃ receptor currents in a non-competitive manner with the mechanism of open channel blocking.

Keyword

Depression; Escitalopram; Patch clamp; Selective serotonin reuptake inhibitor; 5-hydroxytriptamine₃ receptor

MeSH Terms

Antidepressive Agents
Anxiety Disorders
Citalopram*
Depression
Methods
Neuroblastoma
Serotonin*
Antidepressive Agents
Citalopram
Serotonin

Figure

• 

  Fig. 1 Inhibitory effect of escitalopram on 5-hydroxytryptamine (5-HT)3 receptor currents. (A) Representative 5-HT3 receptor currents induced by 3 µM of 5-HT with (black traces) or without (gray trace) escitalopram at different concentrations (1, 3, 10, 30, 100 µM). Open horizontal bar indicates drug application period. 5-HT induced currents were inhibited depending on 5-HT concentration. (B) Concentration-inhibition curve of 5-HT3 receptor current peak amplitudes induced by 3 µM 5-HT co-applied with escitalopram. IC50 of escitalopram on 3 µM 5-HT induced peak amplitudes was 5.35 ± 0.95. Hill slope was −1.28 ± 0.13. Data are expressed as means ± SEM.

• 

  Fig. 2 Concentration-response of 5-hydroxytryptamine (5-HT) induced currents with escitalopram. (A) Representative traces of 5-HT3 receptor currents induced by 0.3, 1, 3, 10, 30 µM of 5-HT in the presence (black traces) or absence (gray traces) of 10 µM escitalopram (ES). Open horizontal bars indicate drug application period. (B) Averaged concentration-response curve of 5-HT3 receptor current peak amplitudes in the presence (closed circle) or absence (open circle) of escitalopram. Escitalopram reduced maximal response of 5-HT induced current *p < 0.001, unpaired t-test) and increased EC50 value of 5-HT without Hill slope change. Data are expressed as means ± SEM.

• 

  Fig. 3 Voltage independency of escitalopram effect. (A) Representative current traces of 3 µM 5-HT at holding potentials of −50, −30, −10, 0, 10, 30 mV without (left) or with (right) escitalopram (ES). Open horizontal bars indicate drug application period. (B) Averaged normalized peak current amplitudes and holding potential (VHolding) relationship with (closed circles) or without (open circles) escitalopram. Peak current amplitudes were decreased in all tested holding potentials. However, reversal potential was not changed by escitalopram. (C) Inhibitory ratio (I5-HT+ES/I5-HT) of 5-HT induced currents by escitalopram at various holding potentials. I5-HT+ES/I5-HT at test holding potential was not significantly different. Data are expressed as means ± SEM.

• 

  Fig. 4 Inhibition of 5-hydroxytryptamine (5-HT)3 receptor currents by escitalopram in different application methods. (A) Representative trace of 5-HT3 receptor current induced by 3 µM 5-HT alone. (B) Representative trace of 5-HT3 receptor current induced by co-application of 5-HT and 10 µM escitalopram (ES). (C) 5-HT3 receptor current trace induced by 5-HT during continuous application of escitalopram from 1 min prior to 5-HT application to the end recording. (D) 5-HT3 receptor current trace induced by 5-HT with pre-application and post-application of escitalopram. At this application method, 5-HT was applied without escitalopram co-application. Open horizontal bars indicate 5-HT application period and closed bars indicate escitalopram application. (E) Comparison of normalized peak amplitudes (IPeak) of 5-HT3 receptor currents among each application mode. Right hatched bar indicates co-application. Filled bar indicates continuous application. Dotted bar indicates pretreatment without co-application of escitalopram. Inhibitory effect by escitalopram was the smallest in the pre-application mode without co-application. *p < 0.001 between indicated pair. Data are expressed as means ± SEM.

• 

  Fig. 5 Association and dissociation kinetics of escitalopram on open 5-hydroxytryptamine (5-HT)3 receptor. (A) Representative superimposed 5-HT3 receptor currents induced by 3 µM 5-HT with 5 sec co-application of escitalopram (ES) at 0 (gray trace), 1, 3, 10, 30, or 100 µM (black traces) 3 sec after the start of 5-HT application. Open horizontal bar indicates 5-HT application period and closed bar indicates escitalopram application. (B) Average of association (open circles) and dissociation (closed circles) time constants (τ) plotted against concentration of escitalopram. (C) Plotting of reverse values of the association time constants (1/τ) against escitalopram concentration. Linear regression fitted to these values and the slope of this function (i.e., an association rate constant) and the y-axis intercept (i.e., a dissociation rate constant) were used to calculate the apparent Kd of escitalopram from equation (4) shown in the Methods. Data are expressed as means ± SEM.

• 

  Fig. 6 Effect of escitalopram on 5-hydroxytryptamine (5-HT)3 receptor desensitization. (A) Representative current traces of 10 sec application of 5-HT with (black trace) or without (gray trace) 10 µM escitalopram. Open horizontal bar indicates drug application period. Current decay during application of 5-HT was accelerated by escitalopram. (B) Comparison of slopes of current decay by 5-HT3 receptor desensitization. Open bar indicates 5-HT alone. Right hatched bar indicates escitalopram co-application. The decay slope was increased by escitalopram. (C, D) Superimposed sample traces evoked by two pulses of 10 µM 5-HT for 5 sec in inter-pulse intervals of 1, 5, 10, 30, 60 sec with (D) or without (C) 10 µM escitalopram. Open arrow-head indicates the first application and filled arrow-head indicates the second application of 5-HT. (E) Averaged data of paired-pulse ratio (the second peak amplitudes/the first peak amplitude) plotted against inter-pulse intervals. A single exponential function was fitted to the data and compared time constants of 5-HT alone (open circle) and co-applied with escitalopram (closed circle). Escitalopram did not change the time course of recovery from desensitization. *p < 0.001. Data are expressed as means ± SEM.

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