HT receptors are distributed throughout the

5-HT3 receptors are distributed throughout the brain, within the brainstem (e.g., nucleus tractus solitarius, area postrema and spinal trigeminal nucleus) and ibotenic acid (e.g., hippocampus, amygdala, nucleus accumbens, putamen and caudate) (Abi-Dargham et al., 1993, Barnes et al., 1989, Bufton et al., 1993, Koyama et al., 2017, Parker et al., 1996). Antagonists for the 5-HT3 receptor, such as ondansetron, granisetron, tropisetron, dolasetron, and palonosetron, are “gold standard” drugs in the treatment of chemotherapy-induced nausea and vomiting (Billio et al., 2010). Moreover, promising data on the therapeutic potential of 5-HT3 receptor antagonists have been reported for the treatment of psychiatric disorders, such as anxiety, depression, schizophrenia, irritable bowel syndrome, cognitive dysfunction, substance abuse and addiction (Walstab et al., 2010). 5-HT3 receptor antagonists are relatively safe but have side effects such as headache, constipation, diarrhea, asthenia, somnolence, and hepatic dysfunction (Goodin and Cunningham, 2002). In addition, the safety of chronic oral dosing of 5-HT3 receptor antagonists has not been defined, suggesting that minimum doses should be administered (Olver et al., 2011). In the present study, we focused on geissoschizine methyl ether, hirsutine, and hirsuteine (which are indole alkaloids) and rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine (which are oxindole alkaloids containing an indole nitrogen) in yokukansan (Ueki et al., 2013) (Fig. 1) and examined their effects on 5-HT3A and 5-HT3AB receptor currents.
Materials and methods
Results Structures of geissoschizine methyl ether, hirsutine, hirsuteine, rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, and 5-HT are shown in Fig. 1 (Ueki et al., 2013). Geissoschizine methyl ether, hirsutine, hirsuteine, and 5-HT are indole alkaloids, and rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine are oxindole alkaloids. To examine the effect of these alkaloids on 5-HT3 receptors, human 5-HT3A or 5-HT3A/5-HT3B subunits were expressed in Xenopus laevis oocytes to create homopentameric 5-HT3A or heteropentameric 5-HT3AB receptors. The seven compounds themselves did not induce any current in 5-HT3A or 5-HT3AB receptors (data not shown), indicating that they were not agonists for 5-HT3A or 5-HT3AB receptors. Next, to examine the modulation of 5-HT-mediated current by these compounds, we simultaneously applied 5-HT at 2 µM [the 20% effective concentration (EC20)], which enabled measurement of the inhibition and potentiation of 5-HT3 receptor current by these compounds. All alkaloidal compounds tested inhibited the 5-HT-mediated 5-HT3A receptor current, although the inhibition was weak compared to that produced by granisetron (IC50 0.0138 µM, pIC50 7.88 ± 0.19 5-HT3A), a 5-HT3 receptor-specific antagonist (Figs. 2, 3 and Table 1). Inhibition of the 5-HT3AB receptor current by most of these alkaloids was almost identical to that of inhibition of the 5-HT3A receptor current except for isocorynoxeine and isorhynchophylline, which inhibited the 5-HT3A receptor current significantly more than the 5-HT3AB receptor current (p < 0.05, Fig. 3 and Table 1). In addition, hirsuteine inhibited the 5-HT3AB receptor current significantly more than the 5-HT3A receptor current (p < 0.05, Fig. 3 and Table 1). Geissoschizine methyl ether did not influence the 5-HT3AB receptor current, showing a half-maximal inhibitory concentration (IC50) of >100 µM. Simultaneous administration of these seven compounds inhibited 5-HT3A and 5-HT3AB receptor currents significantly more than administration of each compound singly (p < 0.05) (Fig. 3H, Table 1). Even isocorynoxeine and hirsutine, which were the strongest inhibitors among the seven alkaloids tested for 5-HT3A and 5-HT3AB receptors, respectively, showed IC50 values of 16.80 and 15.15 µM, respectively. Conversely, simultaneous administration of these seven compounds showed IC50 values of 5.27 and 5.14 µM, respectively (Table 1). These data indicate that these seven yokukansan alkaloids inhibited 5-HT3 receptor current in a cooperative manner.