The 5-Hydroxytryptamine6 Receptor-Selective radioligand [3H]Ro 63–0563 Labels 5-Hydroxytryptamine Receptor Binding Sites in Rat and Porcine Striatum

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Abstract
Ro 63–0563 [4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulfonamide] is a high affinity 5-hydroxytryptamine6 (HT6) receptor antagonist with more than 100-fold selectivity for the 5-HT6 receptor compared with 69 other receptors and binding sites. The present study describes the properties of [3H]Ro 63–0563, the first selective 5-HT6 receptor radioligand. Specific binding of [3H]Ro 63–0563 (nonspecific binding defined in the presence of 10 μm methiothepin) to recombinant rat and human 5-HT6 receptors was saturable, rapid, and reversible with equilibrium dissociation constants (Kd) of 6.8 nmand 4.96 nm, respectively. The pharmacological profile of the rat 5-HT6 receptor labeled with [3H]Ro 63–0563 (methiothepin > d-lysergic acid diethylamide > clozapine ∼ Ro 63–0563 > lisuride > ergotamine ∼ Ro 04–6790 > 5-HT > amitriptyline ∼ metergoline ∼ mianserin ∼ ritanserin > methysergide > mesulergine) was similar to that obtained by using either [3H]d-lysergic acide diethylamide or [3H]5-HT as radioligand. In equilibrium binding studies with rat striatal membranes, [3H]Ro 63–0563 labeled a single binding site with Kd andBmax values of 11.7 nm and 175 fmol/mg protein, respectively. In porcine striatal membranes, [3H]Ro 63–0563 also labeled a single binding site withKd andBmax values of 8.0 nm and 130 fmol/mg protein, respectively. The affinities of 14 5-HT6 receptor ligands at this binding site were similar to those found for the recombinant rat and human 5-HT6receptor, which suggested the presence of 5-HT6 receptors in porcine striatum.