Korean J Physiol Pharmacol.  2019 Sep;23(5):419-426. 10.4196/kjpp.2019.23.5.419.

Gastroprokinetic agent, mosapride inhibits 5-HT₃ receptor currents in NCB-20 cells

Affiliations
  • 1Department of Anatomy, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea.
  • 2Department of Pharmacology, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea. sungkw@catholic.ac.kr

Abstract

Mosapride accelerates gastric emptying by acting on 5-hydroxytryptamine type 4 (5-HT₄) receptor and is frequently used in the treatment of gastrointestinal (GI) disorders requiring gastroprokinetic efficacy. We tested the effect of mosapride on 5-hydroxytryptamine type 3 (5-HT₃) receptor currents because the 5-HT₃ receptors are also known to be expressed in the GI system and have an important role in the regulation of GI functions. Using the whole-cell voltage clamp method, we compared the currents of the 5-HT₃ receptors when 5-HT was applied alone or was co-applied with mosapride in cultured NCB-20 cells known to express 5-HT₃ receptors. The 5-HT₃ receptor current amplitudes were inhibited by mosapride in a concentration-dependent manner. Mosapride blocked the peak currents evoked by the application of 5-HT in a competitive manner because the EC₅₀ shifted to the right without changing the maximal effect. The rise slopes of 5-HT₃ receptor currents were decreased by mosapride. Pre-application of mosapride before co-application, augmented the inhibitory effect of mosapride, which suggests a closed channel blocking mechanism. Mosapride also blocked the opened 5-HT₃ receptor because it inhibited the 5-HT₃ receptor current in the middle of the application of 5-HT. It accelerated desensitization of the 5-HT₃ receptor but did not change the recovery process from the receptor desensitization. There were no voltage-, or use-dependency in its blocking effects. These results suggest that mosapride inhibited the 5-HT₃ receptor through a competitive blocking mechanism probably by binding to the receptor in closed state, which could be involved in the pharmacological effects of mosapride to treat GI disorders.

Keyword

Gastroprokinetic; Mosapride; Patch clamp; 5-hydroxytryptamine3 receptor

MeSH Terms

Gastric Emptying
Methods
Serotonin
Serotonin

Figure

  • Fig. 1 Concentration-dependent inhibition of 5-hydroxytryptamine type 3 (5-HT3) receptor currents by mosapride. (A) Representative current traces induced by 3 µM 5-HT co-applied with 0.3, 1, 3, 10, 30 µM mosapride. The open horizontal bar indicates the drug application period. Mosapride alone did not induce any current even at high concentration (indicated by arrowhead). (B) Averaged concentration-dependent inhibition of mosapride on the 5-HT3 receptor current peak amplitude. The data were normalized values (○) to the peak amplitude induced by 3 µM 5-HT alone and a line was obtained from fitting these data to equation (2) in the Methods. (C) Averaged concentration-dependent block of the 5-HT3 receptor current rise slope by mosapride. Mosapride concentration-dependently decreased the rise slopes of currents at low concentrations (0.3, 1, 3 µM). The data are expressed as means ± standard error of the mean.

  • Fig. 2 Competitive inhibition of mosapride on the 5-hydroxytryptamine type 3 (5-HT3) receptor currents. (A) Representative traces of 5-HT3 receptor currents induced by 1, 3, 10, 30 µM 5-HT in the presence (black traces) or absence of 10 µM of mosapride (gray traces), near IC50 seen in Fig. 1B. The open horizontal bars indicate the drug application period. (B) Averaged concentration-response curve of 5-HT3 receptor currents in the presence (●) or absence (○) of mosapride. The data were normalized to the peak amplitude induced by 10 µM 5-HT and lines were obtained by fitting these data to equation (1) in the Methods. The EC50 of 5-HT was increased from 1.88 ± 0.10 µM (n = 10) to 4.80 ± 0.32 µM (n = 8) by mosapride (p < 0.001, unpaired t-test) without Hill coefficient changes. There were no significant differences in the peak amplitudes induced by 30 µM 5-HT alone and the co-application of mosapride with 30 µM 5-HT (p = 0.7282, unpaired t-test). The data are expressed as means ± standard error of the mean.

  • Fig. 3 Closed channel blocking effect of mosapride on the 5-hydroxytryptamine type 3 (5-HT3) receptor. (A) Representative trace of 5-HT3 receptor currents induced by 5-HT (10 µM) alone, co-application of mosapride (10 µM) and 5-HT, and co-application 1 min after mosapride pretreatment. The open horizontal bar indicates the 5-HT application period and the closed bar indicates mosapride application. (B) Averaged inhibitory effect on the peak amplitude of 5-HT3 receptor currents. Pretreatment for 1 min significantly increased the inhibitory effect of mosapride (n = 10, *p < 0.001, paired t-test). The data are expressed as means ± standard error of the mean.

  • Fig. 4 Effects of mosapride on the open state of 5-hydroxytryptamine type 3 (5-HT3) receptor. (A) Superimposed sample traces show the 5-HT (3 µM, 13 sec, open horizontal bar)-evoked currents and the effect of subsequent application of mosapride co-application (0.3, 1, 3, 10, 30 µM, 5 sec, closed horizontal bar) on these currents. (B) The current decay by mosapride (for 5 sec) 3 sec after 5-HT application was fitted to a single exponential function, and the time constants (τD) were taken as an approximation of the drug-open channel interaction kinetics. The relationship between τD and mosapride concentration was described by equation (3) in the Methods. The slope of this function yielded an association rate constant (k+1 = 0.02 µM−1sec−1) and the intercept at the ordinate gave a dissociation rate constant (k−1 = 0.27 sec−1, n = 9). From equation (4) in the Methods, the calculated IC50 was 12.5 µM. The data are expressed as means ± standard error of the mean.

  • Fig. 5 Effect of mosapride on the desensitization of 5-hydroxytryptamine type 3 (5-HT3) receptor and the time course of desensitization recovery. (A) Representative 5-HT3 receptor currents induced by 10 µM of 5-HT for 10 sec with (black trace) or without (gray trace) 10 µM mosapride. The open horizontal bars indicate the drug application period. (B) Averaged bar graph shows the effects of mosapride on the current decay slope after the long application of 5-HT to test the effect of mosapride on 5-HT3 receptor desensitization (n = 10, *p < 0.01, paired t-test). (C, D) Superimposed sample current traces evoked by two pulses of 10 µM 5-HT for 5 sec in the inter-pulse intervals of 1, 5, 10, 30, 60 sec with (D) or without (C) 10 µM mosapride. Gray arrowheads indicate the first application and the black arrowheads indicate the second application of 5-HT. (E) Averaged data of the paired-pulse ratio (the second peak amplitudes / the first peak amplitude) plotted against the inter-pulse intervals. A single exponential function was fitted to the data and compared to the time constants of 5-HT alone (○, n = 9) and the co-application 5-HT and mosapride (●, n = 9). Mosapride did not change the time course of desensitization recovery of 5-HT3 receptor (p = 0.5588, unpaired t-test). The data are expressed as means ± standard error of the mean.


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