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Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Authors


Address correspondence and reprint requests to Dr. X. Khawaja at Molecular Pharmacology, Wyeth Research (U.K.), Huntercombe Lane South, Taplow, Maidenhead, Berkshire, U.K.

Abstract

Abstract: The specific binding of [3H]WAY-100635 {N-[2-[4-(2-[O-methyl-3H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [3H]WAY-100635 association (k+1 = 0.069 ± 0.015 nM−1 min−1) and dissociation (k−1 = 0.023 ± 0.001 min−1) followed monoexponential kinetics. Saturation binding isotherms of [3H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (Bmax) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [3H]WAY-100635 was ∼36% higher compared with that of 8-hydroxy-2-(di-n-[3H]-propylamino)tetralin ([3H]8-OH-DPAT). The binding affinity of [3H]WAY-100635 was significantly lowered by the divalent cations CaCl2 (2.5-fold; p < 0.02) and MnCl2 (3.6-fold; p < 0.05), with no effect on Bmax. Guanyl nucleotides failed to influence the KD and Bmax parameters of [3H]WAY-100635 binding to 5-HT1A receptors. The pharmacological binding profile of [3H]WAY-100635 was closely correlated with that of [3H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine1A (5-HT1A) sites in rat hippocampus. [3H]WAY-100635 competition curves with 5-HT1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.

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