Clebopride stimulates 5-HT4-serotonin receptors in the human atrium

Figures

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  Fig. 1. Please note how the benzene ring and its substituents were slowly made more complex, from the starting stone namely procainamide (an ancient antiarrhythmic agent) to metoclopramide and clebopride. With arrows, we indicate with which receptors clebopride is known to interact as an agonist or and antagonist or as a partial agonist. Clebopride also inhibits hERG activity, a cardiac potassium channel

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  Fig. 2. Original recording of the time-dependent positive and negative inotropic effects of clebopride on electrically stimulated human right atrial muscle strips, demonstrating its dual action as an agonist (A) or antagonist (C). To compare the efficacy of different compounds, cilostamide (Cilo, 100 nM), clebopride (Clebo, 10 µM), serotonin (Sero, 1 µM), and finally isoprenaline (Iso, 1 µM) were sequentially added to the organ bath (A). In a separate series of experiments (C), serotonin (Sero, 1 µM) was added first, followed by clebopride (Clebo, 10 µM), to evaluate its antagonistic effect. To verify the veracity of this negative inotropic effect, isoprenaline (Iso, 1 µM) was added last. In (A) and (C), horizontal lines represent time (in minutes), while vertical lines indicate the developed force of contraction (in milli Newton, mN). The summarized effects of these drugs are presented in Figure 2B and D. The ordinate axis in (B) represents the force of contraction in mN, whereas in Figure 2D, it is expressed as a percentage (%) of the pre-drug value (control, CTR). The number of experiments is indicated within the bars. * denote statistically significant differences (*p < 0.05) based…

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  Figure 3. A Original recording of the time-dependent positive inotropic effect of clebopride (measured in milli Newton, mN) in electrically stimulated human right atrial muscle strips. Cilostamide (Cilo, 100 nM) was added first, followed by increasing concentrations of clebopride (Clebo, 0.1–10 µM), and finally the 5-HT₄ serotonin receptor antagonist GR125487 (GR, 1 µM). These data are summarized in the bar diagram in B. Moreover, clebopride (Clebo, 0.1–10 µM) was applied either alone (C, bottom) or in combination with cilostamide (Cilo, 100 nM; C, top). The data from experiments such as those shown in C are summarized in D, indicating that clebopride alone does not increase contractile force but does so in the presence of the phosphodiesterase III inhibitor cilostamide. In A and C, horizontal lines represent time (in minutes), while vertical lines indicate developed force of contraction (in mN). In B and D, the ordinate axis represents the force of contraction as a percentage of the pre-drug value (Cilo, 100 nM). The number of experiments is indicated within the bars. * denote statistically significant differences (*p < 0.05) based on two-way ANOVA followed by Bonferroni’s multiple…

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  Fig. 4. In samples such as those shown in Figure 2, we measured the effects of clebopride on contractile parameters. Cilostamide (Cilo, 100 nM) was administered first, followed by clebopride (Clebo, 10 µM), serotonin (Sero, 1 µM), and finally isoprenaline (Iso, 1 µM; Figure 2A). Data on time to peak tension (T1) and time of relaxation (T2) in ms are presented in A, while data on the rate of tension development (+dF/dt) and rate of relaxation (-dF/dt) in milli Newtons per second (mN/s) under the same conditions are shown in B. “n” indicates number of experiments. Asterisks (*) indicate significant differences (p < 0.05) compared to pre-drug values (CTR). # denote significant differences (p < 0.05) between the values indicated by the dotted lines. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison post hoc test

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  Fig. 5. Original recordings of isolated, electrically stimulated mouse left atrial preparations (LA) from 5-HT₄-TG (lower trace) and WT (upper trace) are shown in A. Clebopride, administered cumulatively from 0.1 nM to 10 µM, induced a time- and concentration-dependent positive inotropic effect in 5-HT₄-TG. B illustrates that the positive inotropic effect of clebopride is selectively mediated via the 5-HT₄ receptor, occurring only in TG (age: 151.5 ± 68.3 days; ♀: 4) and not in WT (age: 97.8 ± 4 days; ♂: 4). The vertical axis represents the contraction force in milli Newtons (mN), while the horizontal axis indicates clebopride concentration in negative decadic molar values (-Log [M]). C (original recording) shows that the positive inotropic effect of clebopride is antagonized by GR125487 (GR, 0.1 µM). Several datasets similar to those in Figure 2C are summarized in D, demonstrating that clebopride (Clebo, 1 µM) induces a time- and concentration-dependent positive inotropic effect in 5-HT₄-TG, which is effectively antagonized by GR125487 (GR, 0.1 µM). The number of experiments conducted is indicated within the bars of the graph. * denote significant differences (p < 0.05) compared to the…

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  Fig. 6. Original recordings of the effects of clebopride on right atrial (RA) preparations from 5-HT₄-TG (lower trace) and WT (upper trace) are shown in A. Clebopride induced a time- and concentration-dependent positive chronotropic effect in isolated, spontaneously beating RA preparations from 5-HT₄-TG, but not in RA from WT. This is summarized in B. In A and B, the ordinates represent beating rates in beats per minute (BPM), and the horizontal lines indicate clebopride concentrations in negative decadic molar units (-Log [M]). The positive chronotropic effects of clebopride are antagonized by the 5-HT₄ receptor antagonist GR125487 (GR; 0.1 µM), as shown in the original recordings in C. The summarized data from these experiments are presented in D. It is important to note that data were collected from 5-HT₄-TG (age: 212.5 ± 109.7 days; ♀: 6), and, for direct comparison, data from WT (age: 148.3 ± 115.3 days; ♂: 5, ♀: 1) were also included. The number of experiments conducted is indicated within the bars of the graph. The ordinates represent beats per minute (BPM). The abscissa in C shows clebopride concentrations in negative decadic molar units, whereas D presents the drug…

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  Fig. 7. As shown in Figure 5, the positive inotropic effect of clebopride (Clebo, 1–10 µM) occurs exclusively in left atrial (LA) preparations from 5-HT₄-TG (age: 151.5 ± 68.3 days; ♀: 4), whereas this effect is absent in WT (age: 97.8 ± 4 days; ♂: 4). The 5-HT₄ receptor antagonist GR125487 (GR, 0.1 µM) abolishes this increase in contractile force. Data on time to peak tension (T1) and relaxation time (T2) in ms are presented in A and B, respectively, while data on the rate of tension development (+dF/dt) and relaxation (-dF/dt) in milli Newtons per second (mN/s) under the same conditions are shown in C. The number of experiments is indicated in parentheses followed by “n” in each case