Vol. 288, Issue 1, 221-231, January 1999

Characterization of (S)-Des-4-amino-3-[¹²⁵I]iodozacopride ([¹²⁵I]DAIZAC), a Selective High-Affinity Radioligand for 5-Hydroxytryptamine₃ Receptors¹

William A. Hewlett, Bakula L. Trivedi, Zhang-Jin Zhang, Tomas de Paulis, Dennis E. Schmidt, David M. Lovinger, M. Sib Ansari and Michael H. Ebert

Departments of Psychiatry, Radiology, and Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee

The 5-hydroxytryptamine(HT)₃ receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2.2]oct-3-yl)benzamide] is a compound with high affinity and selectivity 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 in rat brain (K_D = 0.15 nM). Nonspecific binding of [¹²⁵I]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 µM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT₃ receptor over the M₁ muscarinic binding site, 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 µM) were ineffective in displacing [¹²⁵I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3-30 µM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [¹²⁵I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT₃ receptor-mediated inward cation current in NCB-20 cells with an IC₅₀ of 0.24 nM. [¹²⁵I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT₃ receptor.