title: Characteristics of binding of [3H]WAY100635 to rat hippocampal membranes.
abstract: Kinetic analysis of binding of [(3)H][N-[2-[4-(2-[O-methyl-(3)H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide ([(3)H]WAY100635) to 5-HT(1A) receptors in rat hippocampal membranes has revealed complex regulation mechanism for this radioligand. Saturation binding experiments revealed that [(3)H]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) x 10(-3) min(-1), means that equilibrium with this radioligand cannot be achieved before 7.5 h incubation at 25 degrees C. Systematic association kinetic studies of [(3)H]WAY100635 binding revealed sharp reaction acceleration at higher radioligand concentration, proposing mechanism of positive cooperativity. The affinities of antagonists determined from competition with [(3)H]WAY100635 did not coincide with their abilities to inhibit 5-HT-dependent activation of [(35)S]GTPgammaS binding probably due to the ligand's kinetic peculiarities. Thus, [(3)H]WAY100635 appears to be an excellent tool for determining receptor binding sites, but its applicability in equilibrium studies is strongly limited.
authors.affiliation: Institute of Organic and Bioorganic Chemistry, University of Tartu, Jakobi Str. 2, 51014, Tartu, Estonia. ago.rinken@ut.ee
authors.forename: Sven
authors.initials: S
authors.lastname: Parkel
authors.forename: Ago
authors.initials: A
authors.lastname: Rinken
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology*; D010879:Piperazines / Q000493:pharmacokinetics*; D011725:Pyridines / Q000493:pharmacokinetics*; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D044282:Receptor, Serotonin, 5-HT1A / Q000378:metabolism; D012702:Serotonin Antagonists / Q000493:pharmacokinetics*; D014316:Tritium / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D010879:Piperazines; D011725:Pyridines; D012702:Serotonin Antagonists; D014316:Tritium; D044282:Receptor, Serotonin, 5-HT1A; C058895:1-(2-methoxyphenyl)-4-(4-(2-phthalimido)butyl)piperazine; C081293:WAY 100135; C090413:N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide