In the present study, binding affinities of 5-hydroxytryptamine-4 (5-HT
4) ligands for the human 5-HT
4d receptor were determined using the agonist [
3H]5-HT and the selective 5-HT
4 antagonist [
3H]GR113,808. We also compared the affinity differences between [
3H]5-HT binding (K
H) and [
3H]GR113,808 binding (K
L) with their activities as 5-HT
4 ligands. Binding studies using [
3H]5-HT revealed that the human 5-HT
4d receptor has two binding sites, whereas [
3H]GR113,808 yielded a single binding site. Additionally, the number of [
3H]5-HT binding sites decreased in the presence of guanosine-5'-
O-(3-thiotriphosphate) (GTPγS), but the number of [
3H]GR113,808 sites did not change. In competitive binding assays, full agonists such as 5-methoxytryptamine and tegaserod showed 2- to 8-fold higher affinities for [
3H]5-HT binding (K
H) than for [
3H]GR113,808 binding (K
L) (K
H<K
L). Conversely, antagonists showed lower affinities for [
3H]5-HT binding than for [
3H]GR113,808 binding (K
H>K
L). Finally, partial agonists displayed similar binding affinities for both radioligands (K
H = K
L). These findings suggest that the equilibrium between active and inactive states of the human 5-HT
4d receptor relies on the functional activities of 5-HT
4 ligands, and these states affect the affinities of 5-HT
4 ligands in the competitive binding assay.
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