title: Characterisation of the binding of [3H]WAY-100635, a novel 5-hydroxytryptamine1A receptor antagonist, to rat brain.
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 approximately 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 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), 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 GTP gamma S, indicating the existence of GTP-insensitive sites.
authors.affiliation: Department of Molecular Pharmacology, Wyeth Research (U.K.), Maidenhead, Berkshire, England.
authors.forename: X
authors.initials: X
authors.lastname: Khawaja
authors.forename: N
authors.initials: N
authors.lastname: Evans
authors.forename: Y
authors.initials: Y
authors.lastname: Reilly
authors.forename: C
authors.initials: C
authors.lastname: Ennis
authors.forename: M C
authors.initials: MC
authors.lastname: Minchin
mesh_terms: D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008297:Male; D010879:Piperazines / Q000378:metabolism*; D011725:Pyridines / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012702:Serotonin Antagonists / Q000378:metabolism*; D013696:Temperature; D014316:Tritium
chemical_list: D002413:Cations, Divalent; D002414:Cations, Monovalent; D010879:Piperazines; D011725:Pyridines; D012702:Serotonin Antagonists; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C090413:N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin