CHARACTERIZATION OF THE BINDING OF [H-3] WAY-100635, A NOVEL 5-HYDROXYTRYPTAMINE(1A) RECEPTOR ANTAGONIST, TO RAT-BRAIN

The specific binding of [H-3]WAY-100635 {N-[2- ethoxyphenyl)-1-piperaz inyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and ti ssue concentration dependent. The rates of [H-3]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 b inding isotherms of [H-3]WAY-100635 exhibited a single class of recogn ition site with an affinity of 0.37 +/- 0.051 nM and a maximal binding capacity (B-max) of 312 +/- 12 fmol/mg of protein, The maximal number of binding sites labelled by [H-3]WAY-100635 was similar to 36% highe r compared with that of 8-hydroxy-2-(di-n-[H-3]-propylamino)tetralin ( [H-3]8-OH-DPAT). The binding affinity of [3H]WAY-100635 was significan tly lowered by the divalent cations CaCl2 (2.5-fold; p < 0.02) and MnC l2 (3.6-fold; p < 0.05), with no effect on B-max. Guanyl nucleotides f ailed to influence the K-D and B-max parameters of [H-3]WAY-100635 bin ding to 5-HT1A receptors, The pharmacological binding profile of [H-3] WAY-100635 was closely correlated with that of [H-3]8-OH-DPAT, which i s consistent with the labelling of 5-hydroxytryptamine(1A) (5HT(1A)) s ites in rat hippocampus. [H-3]WAY-100635 competition curves with 5-HT1 A agonists and partial agonists were best resolved into high- and low- affinity binding components, whereas antagonists were best described b y a one-site binding model. In the presence of 50 mu M guanosine 5'-O- (3-thiotriphosphate) (GTP gamma S), competition curves for the antagon ists remained unaltered, whereas the agonist and partial agonist curve s were shifted to the right, reflecting an influence of G protein coup ling on agonist versus antagonist binding to the 5-HT1A receptor, Howe ver, a residual (16 +/- 2%) high-affinity agonist binding component wa s still apparent in the presence of GTP gamma S, indicating the existe nce of GTP-insensitive sites.