Characteristics of Binding of [3H]WAY100635 to Rat Hippocampal Membranes |
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Authors: | Sven Parkel Ago Rinken |
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Affiliation: | (1) Institute of Organic and Bioorganic Chemistry, University of Tartu, Jakobi Str. 2, 51014 Tartu, Estonia |
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Abstract: | Kinetic analysis of binding of [3H][N-[2-[4-(2-[O-methyl-3H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide ([3H]WAY100635) to 5-HT1A receptors in rat hippocampal membranes has revealed complex regulation mechanism for this radioligand. Saturation binding experiments revealed that [3H]WAY100635 binds to a single class of receptors with very high apparent affinity (K D = 87 ± 4 pM, B max = 15.1 ± 0.2 fmol/mg protein). The binding was almost irreversible, as the dissociation rate constant obtained k off = (7.8 ± 1.1) × 10−3 min−1, means that equilibrium with this radioligand cannot be achieved before 7.5 h incubation at 25°C. Systematic association kinetic studies of [3H]WAY100635 binding revealed sharp reaction acceleration at higher radioligand concentration, proposing mechanism of positive cooperativity. The affinities of antagonists determined from competition with [3H]WAY100635 did not coincide with their abilities to inhibit 5-HT-dependent activation of [35S]GTPγS binding probably due to the ligand’s kinetic peculiarities. Thus, [3H]WAY100635 appears to be an excellent tool for determining receptor binding sites, but its applicability in equilibrium studies is strongly limited. |
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Keywords: | [3H]WAY100635 5-HT1A receptor Rat Hippocampus Kinetics Saturation binding Serotonergic antagonist |
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