Agonists and antagonists induce different palonosetron dissociation rates in 5-HT₃A and 5-HT₃AB receptors

Abstract

Palonosetron is a potent 5-HT₃ receptor antagonist with a unique structure and some unusual properties. Here we explore the properties of palonosetron at heterologously expressed 5-HT₃A and 5-HT₃AB receptors. We used receptors expressed in HEK293 cells, and functionally analysed them using a membrane potential sensitive dye in a Flexstation, which revealed IC₅₀s of 0.24 nM and 0.18 nM for 5-HT₃A and 5-HT₃AB receptors respectively. Radioligand binding studies with [(3)H]palonosetron revealed similar Kds: 0.34 nM for 5-HT3A and 0.15 nM for 5-HT₃AB receptors. Kinetic studies showed palonosetron association and dissociation rates were slightly faster in 5-HT₃AB than 5-HT₃A receptors, and for both subtypes dissociation rates were ligand-dependent, with antagonists causing more rapid dissociation than agonists. Similar ligand effects were not observed for [(3)H]granisetron dissociation studies. These data support previous studies which show palonosetron has actions distinct to other 5-HT3 receptor antagonists, and the slow rates observed for agonist induced dissociation (t₁/₂ > 10 h) could at least partly explain the long duration of palonosetron effects in vivo.

Keywords: 5-HT; 5-hydroxytryptamine (serotonin); Allosteric binding site; FlexStation assays; HEK; IC(50); K(d); Radioligand binding; Serotonin receptor; Site-directed mutagenesis; affinity constant; concentration of antagonist required for half-maximal inhibition; human embryonic kidney.

Fig. 1

Chemical structures of 5-HT and clinically used 5-HT₃ receptor competitive antagonists.

Fig. 2

Typical Flexstation responses of HEK293 cells expressing 5-HT₃A and 5-HT₃AB receptors. 5-HT at various concentrations (0–30 μM) was added at 20 s. Note the shapes of the responses, which are different in homomeric and heteromeric receptors. F = arbitrary fluorescent units.

Fig. 3

Inhibition of 5-HT-induced responses in HEK293 cells. A: Palonosetron is more potent when pre-applied () than when co-applied () with 5-HT; example in cells expressing 5-HT₃A receptors. B. Palonosetron has similar IC₅₀s at 5-HT₃A and 5-HT₃AB receptors. Parameters derived from these data are given in Section 3.1.

Fig. 4

Functional and radioligand binding data suggest similar effects of palonosetron at 5-HT₃A and 5-HT₃AB receptors. Top: Typical Flexstation data from 5-HT₃A and 5-HT₃AB receptor-expressing cells; EC₅₀ values increase and F_max values decrease as [palonosetron] increases. In this typical example EC₅₀s are 0.16 μM, 0.35 μM and 0.97 μM with relative F_max values of 100%, 70% and 40% for 0, 0.1 nM and 0.3 nM palonosetron in 5-HT₃A receptors, and 0.12 μM (100%), 0.27 μM (62%) and 1.6 μM (28%) in 5-HT₃AB receptors. Lower panel: typical radioligand binding curves for 5-HT₃A and 5-HT₃AB receptors.

Fig. 5

Association and dissociation curves for [³H]palonosetron using HEK293 cells expressing 5-HT₃A and 5-HT₃AB receptors. Association was rapid for both 5-HT₃A and 5-HT₃AB receptors, with maximal levels being reached within 30 min. Dissociation rates were slower, although were faster with antagonists (MDL72222, palonosetron) than agonists (quipazine). Parameters derived from these data are given in Section 3.3.

Fig. 6

[³H]granisetron and [³H]palonosetron dissociation curves in 5-HT₃A receptors. Dissociation curves for [³H]granisetron using HEK293 cells expressing 5-HT₃A receptors using 5-HT (agonist) or MDL72222 (antagonist) are superimposable. However data from [³H]palonosetron dissociation experiments reveal a clear distinction between agonist and antagonist displacement rates. Data from these curves are shown in Table 1.