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.
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