Haloperidol, a typical antipsychotic, inhibits 5-HT₃ receptor-mediated currents in NCB-20 cells: a whole-cell patch-clamp study

Figures

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  Fig. 1. Inhibition of 5-hydroxytryptamine (5-HT)3 receptor-mediated currents by haloperidol. (A) Representative whole-cell current traces recorded from NCB-20 cells expressing 5-HT3 receptors. Currents were induced by 3 µM 5-HT alone (bottom trace) or in the presence of increasing concentrations (1, 3, 10, 30, 100 µM) of haloperidol. As the concentration of haloperidol increased, there was a progressive reduction in the peak amplitude of the 5-HT3 receptor currents, indicating a concentration-dependent inhibition. (B) Dose-response curve for haloperidol’s effect on normalized peak amplitude of the 5-HT3 receptor currents, with currents normalized to those induced by 3 µM 5-HT alone. (C) Concentration-response curve for the effect of haloperidol on normalized rise slope of the 5-HT3 receptor currents. Data are expressed as mean ± SEM.

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  Fig. 2. Modulation of 5-hydroxytryptamine (5-HT)3 receptor currents by haloperidol. (A) Representative whole-cell current traces from NCB-20 cells in response to 1, 3, 10, and 30 µM 5-HT (gray traces) alone or along with the presence of 10 µM haloperidol (black traces). (B) Representative whole-cell current traces as in (A), but with 30 µM haloperidol. Haloperidol inhibits 5-HT-induced currents in a concentration-dependent manner. (C) Averaged concentration-response curves for 5-HT in the absence (open circles) and presence of 10 µM (closed circles) and 30 µM (closed box) haloperidol. The presence of haloperidol caused a rightward shift in the EC50 of 5-HT and a significant reduction in the maximal response (Emax) at 30 µM, suggesting both a decrease in potency and efficacy. Data are expressed as mean ± SEM.

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  Fig. 3. Effect of haloperidol on voltage-current relationship of 5-hydroxytryptamine (5-HT)3 receptor-mediated currents. (A) Representative whole-cell current traces of 5-HT3 receptor currents obtained at various holding potentials (–50, –30, –10, +10, +30 mV). The left set of traces shows currents induced by 3 µM 5-HT alone, while the right set of traces shows currents in the presence of 10 µM haloperidol co-applied with 3 µM 5-HT. Haloperidol reduced the amplitude of 5-HT-induced currents across all holding potentials. (B) Averaged data of normalized peak amplitude of 5-HT3 receptor currents as a function of holding potential. Open circles represent data from 3 µM 5-HT alone, and closed circles represent data from 3 µM 5-HT with 10 µM haloperidol. The reversal potential (VRes) was 3.31 ± 0.33 mV for 5-HT alone and 5.22 ± 0.88 mV for 5-HT co-applied with haloperidol. There was no statistically significant difference in reversal potentials between the two conditions (p = 0.0867, paired t-test, n = 13). Data are expressed as mean ± SEM.

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  Fig. 4. Effects of haloperidol on deactivation and desensitization kinetics of 5-hydroxytryptamine (5-HT)3 receptor-mediated currents. (A) Deactivation was analyzed by applying 10 µM 5-HT for 35 msec and measuring the decay phase of the current. The left side shows representative current traces for 5-HT alone (gray trace) and for 5-HT co-applied with 10 µM haloperidol (black trace). The right side shows decay time constants (τ) for each condition, with open circles representing 5-HT alone and closed circles representing 5-HT with haloperidol. The average decay τ was significantly reduced from 1,521 ± 98.68 msec for 5-HT alone to 1,437 ± 95.82 msec for co-application with haloperidol (paired t-test, p < 0.001, n = 11), indicating an accelerated deactivation process. (B) Desensitization assessed by applying 10 µM 5-HT for 20 sec and analyzing the decay phase. The left side shows representative current traces under both conditions, and the right graph displays the decay time constants, with open circles for 5-HT alone and closed circles for the combination with haloperidol. The average decay τ decreased from 2,222 ± 150.8 msec to 1,869 ± 124.6 msec with haloperidol (paired t-test, p <…

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  Fig. 5. Effects of haloperidol on recovery from deactivation and desensitization of 5-hydroxytryptamine (5-HT)3 receptor-mediated currents. (A) Recovery from deactivation was assessed using a two-pulse protocol, with a first pulse of 10 µM 5-HT applied for 35 msec (gray arrow) followed by a second pulse of 2.5 sec at varying inter-pulse intervals (5, 10, 20, 30, 60 sec; black arrows). The left trace shows representative currents for 5-HT alone. The middle trace shows currents for 5-HT co-applied with 10 µM haloperidol. The right graph shows a paired pulse ratio (second peak amplitude/first peak amplitude) as a function of inter-pulse interval. Open circles represent 5-HT alone, and closed circles represent 5-HT with haloperidol. Time constants (τ) for recovery were obtained by fitting paired pulse ratios to a one-phase association equation, yielding a value of 3.45 ± 0.20 sec (n = 9) for 5-HT alone and 3.51 ± 0.22 sec (n = 10) for the co-application (unpaired t-test, p = 0.8450). (B) Recovery from desensitization analyzed with a similar protocol, but with a longer first pulse duration of 5 sec (gray bar). The left trace shows representative currents for 5-HT alone. The middle trace shows…

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  Fig. 6. Use-dependency of haloperidol on 5-hydroxytryptamine (5-HT)3 receptor currents in NCB-20 cells. (A) Representative current traces recorded during the application of twenty 1-sec pulses of 10 µM 5-HT at 30-sec intervals. Upper traces show the control condition with 5-HT alone, while lower traces display the test condition with 10 µM haloperidol co-applied with 5-HT from the 6th pulse (2.5 min) to the 10th pulse (4.5 min), indicated by grey traces. Bars above traces indicate application of drug. (B) Averaged normalized peak amplitude of 5-HT3 receptor currents plotted against time for both control (n = 10, open circles) and test groups (n = 11, closed circles). A gradual decrease in current amplitude was observed in both groups, likely due to current rundown. (C) Comparison of the peak amplitude ratio (10th/6th pulse) between control and test groups to assess use-dependent inhibition. The bar graph shows no significant difference in peak amplitude ratio between 5-HT alone (0.98 ± 0.01) and haloperidol co-application (0.98 ± 0.01), with a p-value of 0.5702 (unpaired t-test). These findings indicated that haloperidol did not induce significant use-dependent inhibition of 5-HT3…

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  Fig. 7. Comparison of pretreatment and co-application effects of haloperidol on 5-hydroxytryptamine (5-HT)3 receptor currents. (A) Representative current traces recorded under four different experimental conditions: from left, 3 µM 5-HT alone, co-application of 3 µM 5-HT with 10 µM haloperidol, pretreatment with 10 µM haloperidol for 3 sec followed by 3 µM 5-HT alone, and pretreatment with 10 µM haloperidol for 3 sec followed by co-application with 3 µM 5-HT. All haloperidol treatments reduced peak amplitude of 5-HT3 receptor currents compared to 5-HT alone. (B) Averaged normalized peak amplitudes for the three haloperidol conditions, normalized to the peak amplitude of 5-HT alone. Co-application of 5-HT and haloperidol (blank bar) resulted in a normalized peak amplitude of 0.73 ± 0.04, while pretreatment with haloperidol followed by 5-HT alone (doted bar) resulted in a normalized peak amplitude of 0.74 ± 0.02. Pretreatment with haloperidol followed by co-application with 5-HT (hatched bar) resulted in the greatest inhibition, with a normalized peak amplitude of 0.54 ± 0.03. The difference in peak amplitude between co-application and haloperidol pretreatment followed by 5-HT alone was…