Characterization of (S)-des-4-amino-3-[I-125]iodozacopride ([I-125]DAIZAC), a selective high-affinity radioligand for 5-hydroxytryptamine(3) receptors

The 5-hydroxytryptamine(HT)(3) receptor subtype is present in the central n ervous system (CNS) in low abundance, and few selective radiolabeled antago nists with high specific activity are available to study these sites. DAIZA C [desamino-3-iodo-(S)zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicy clo-[2.2.2]oct-3-yl)benzamide] is a compound with high affinity and selecti vity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB- 20 cell membranes gave a B-max of 340 +/- 58 fmol/mg protein and a K-D of 0 .14 +/- 0.03 nM, which is in agreement with the value previously reported i n rat brain (K-D = 0.15 nM). Nonspecific binding of [I-125]DAIZAC in NCB-20 cells was <1% of total binding at the K-D for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 mu M) showed minimal abilit y to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discriminatio n ratio of DAIZAC for the 5-HT3 receptor over the M-1 muscarinic binding si te, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3-30 mu M ) were ineffective in displacing [I-125]DAIZAC binding in rat brain membran es. Similarly, antagonists (3-30 mu M) for other nonserotonergic receptors and uptake sites were ineffective in displacing [I-125]DAIZAC binding. Auto radiographic studies showed highest specific binding in area postrema and n ucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation cu rrent in NCB-20 cells with an IC50 of 0.24 nM. [I-125]DAIZAC is a potent an d highly selective ligand for in vitro studies of the 5-HT3 receptor.